US20040101886A1 - Nested oligonucleotides containing a hairpin for nucleic acid amplification - Google Patents
Nested oligonucleotides containing a hairpin for nucleic acid amplification Download PDFInfo
- Publication number
- US20040101886A1 US20040101886A1 US10/628,109 US62810903A US2004101886A1 US 20040101886 A1 US20040101886 A1 US 20040101886A1 US 62810903 A US62810903 A US 62810903A US 2004101886 A1 US2004101886 A1 US 2004101886A1
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- US
- United States
- Prior art keywords
- primer
- ser
- artificial sequence
- seq
- oligonucleotide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Definitions
- This disclosure relates to engineered templates useful for amplification of a target nucleic acid sequence. More specifically, templates which are engineered to contain a predetermined sequence and a hairpin structure are provided by a nested oligonucleotide extension reaction.
- the engineered templates allow Single Primer Amplification (SPA) to amplify a target sequence within the engineered template.
- SPA Single Primer Amplification
- the target sequences from the engineered templates are cloned into expression vehicles to provide a library of polypeptides or proteins, such as, for example, an antibody library.
- nucleic acid amplification and detection of amplification products assist in the detection, identification, quantification, isolation and sequence analysis of nucleic acid sequences.
- Nucleic acid amplification is an important step in the construction of libraries from related genes such as, for example, antibodies. These libraries can be screened for antibodies having specific, desirable activities.
- Nucleic acid analysis is important for detection and identification of pathogens, detection of gene alteration leading to defined phenotypes, diagnosis of genetic diseases or the susceptibility to a disease, assessment of gene expression in development, disease and in response to defined stimuli, as well as the various genome projects.
- Other applications of nucleic acid amplification method include the detection of rare cells, detection of pathogens, and the detection of altered gene expression in malignancy, and the like.
- Nucleic acid amplification is also useful for qualitative analysis (such as, for example, the detection of the presence of defined nucleic acid sequences) and quantification of defined gene sequences (useful, for example, in assessment of the amount of pathogenic sequences as well as the determination of gene multiplication or deletion, and cell transformation from normal to malignant cell type, etc.).
- the detection of sequence alterations in a nucleic acid sequence is important for the detection of mutant genotypes, as relevant for genetic analysis, the detection of mutations leading to drug resistance, pharmacogenomics, etc.
- nucleic acid amplification for example, exponential amplification, linked linear amplification, ligation-based amplification, and transcription-based amplification.
- exponential nucleic acid amplification method is polymerase chain reaction (PCR) which has been disclosed in numerous publications. See, for example, Mullis et al. Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Mullis K. EP 201,184; Mullis et al. U.S. Pat. No. 4,582,788; Erlich et al. EP 50,424, EP 84,796, EP 258,017, EP 237,362; and Saiki R. et al. U.S. Pat. No.
- PCR polymerase chain reaction
- Amplification methods that employ a single primer have also been disclosed. See, for example, U.S. Pat. Nos. 5,508,178; 5,595,891; 5,683,879; 5,130,238; and 5,679,512.
- the primer can be a DNA/RNA chimeric primer, as disclosed in U.S. Pat. No. 5,744,308.
- TSOs template switching oligonucleotides
- blocking oligonucleotides blocking oligonucleotides
- TSOs template switching oligonucleotides
- a template switch amplification in which chimeric DNA primer are utilized is disclosed in U.S. Pat. Nos. 5,679,512; 5,962,272; 6,251,639; and by Patel et al. Proc. Natl. Acad. Sci. U.S.A. 93:2969-2974 (1996).
- the transcription base amplification methods such as Nucleic Acid Sequence Based Amplification (NASBA) and transcription mediated amplification (TMA)
- NASBA Nucleic Acid Sequence Based Amplification
- TMA transcription mediated amplification
- NASBA Nucleic Acid Sequence Based Amplification
- TMA transcription mediated amplification
- Another example of a drawback of the current amplification methods is the requirement of two binding events which may have optimal binding at different temperatures. This combination of factors results in increased likelihood of mis-priming and resultant amplification of sequences other than the target sequence. Therefore, there is a need for improved nucleic acid amplification methods that overcome these drawbacks.
- the invention provided herein fulfills this need and provides additional benefits.
- a method of amplifying nucleic acid which includes the steps of a) annealing a primer to a template nucleic acid sequence, the primer having a first portion which anneals to the template and a second portion of predetermined sequence; b) synthesizing a polynucleotide that anneals to and is complementary to the portion of the template between the location at which the first portion of the primer anneals to the template and the end of the template, the polynucleotide having a first end and a second end, wherein the first end incorporates the primer; c) separating the polynucleotide synthesized in step (b) from the template; d) annealing a nested oligonucleotide to the second end of the polynucleotide synthesized in step (b), the nested oligonucleotide having a first portion that anneals to the second end of the polynucleotide, and a second portion
- the method of amplifying nucleic acid includes the steps of a) annealing a primer and a boundary oligonucleotide to a template nucleic acid sequence, the primer having a first portion which anneals to the template and a second portion of predetermined sequence; b) synthesizing a polynucleotide that anneals to and is complementary to the portion of the template between the location at which the first portion of the primer anneals to the template and the portion of the template to which the boundary oligonucleotide anneals, the polynucleotide having a first end and a second end, wherein the first end incorporates the primer; c) separating the polynucleotide synthesized in step (b) from the template; d) annealing a nested oligonucleotide to the second end of the polynucleotide synthesized in step (b), the nested oligonucleotide
- the method of amplifying nucleic acid includes the steps of a) annealing an oligo dT primer and a boundary oligonucleotide to an mRNA template; b) synthesizing a polynucleotide that anneals to and is complementary to the portion of the template between the location at which the first portion of the primer anneals to the template and the portion of the template to which the boundary oligonucleotide anneals, the polynucleotide having a first end and a second end, wherein the first end incorporates the primer; c) separating the polynucleotide synthesized in step (b) from the template; d) annealing a nested oligonucleotide to the second end of the polynucleotide synthesized in step (b), the nested oligonucleotide having a first portion that anneals to the second end of the polynucleotide
- an engineered nucleic acid strand which has a predetermined sequence at a first end thereof, a sequence complementary to the predetermined sequence at the other end thereof, and a hairpin structure therebetween.
- a method of amplifying a nucleic acid strand which includes the steps of providing an engineered nucleic acid strand having a predetermined sequence at a first end thereof, a sequence complementary to the predetermined sequence at the other end thereof and a hairpin structure therebetween, and contacting the engineered nucleic acid strand with a primer containing at least a portion of the predetermined sequence in the presence of a polymerase and nucleotides under conditions suitable for polymerization of the nucleotides.
- restriction sites can be used to digest the strand so that the target nucleic acid sequence can be ligated into a suitable expression vector.
- the vector may then be used to transform an appropriate host organism using standard methods to produce the polypeptide or protein encoded by the target sequence.
- the techniques described herein are used to amplify a family of related sequences to build a complex library, such as, for example, an antibody library.
- FIG. 1 is a schematic illustration of a primer and boundary oligo annealed to a template
- FIG. 2A is a schematic illustration of a restriction oligo annealed to a nucleic acid strand
- FIG. 2B is a schematic illustration of a primer annealed to a template that has a shortened 5′ end
- FIG. 3 is a schematic illustration of a nested oligo having a hairpin structure annealed to a newly synthesized nucleic acid strand;
- FIG. 4A is a schematic illustration of an engineered template in accordance with this disclosure.
- FIG. 4B is a schematic illustration of an engineered template in accordance with an alternative embodiment.
- FIGS. 5 A- 5 C is a chart showing the sequences of clones produced in Example 4.
- FIGS. 6 A- 6 C is a chart showing the sequences of clones produced in Example 7.
- the present disclosure provides a method of amplifying a target nucleic acid sequence.
- the target nucleic acid sequence is a gene encoding a polypeptide or protein.
- the disclosure also describes how the products of the amplification may be cloned and expressed in suitable expression systems.
- the techniques described herein are used to amplify a family of related sequences to build a complex library, such as, for example, an antibody library.
- the target nucleic acid sequence is exponentially amplified through a process that involves only a single primer.
- the ability to employ a single primer is achieved by engineering a strand of nucleic acid that contains the target sequence to be amplified.
- the engineered strand of nucleic acid (sometimes referred to herein as the “engineered template”) is prepared from two templates; namely, 1) a starting material that is a natural or synthetic nucleic acid (e.g., RNA, DNA or cDNA) containing the sequence to be amplified and 2) a nested oligonucleotide that provides a hairpin structure.
- the starting material can be used directly as the original template, or, alternatively a strand complementary to the starting material can be prepared and used as the original template.
- the nested oligonucleotide is used as a template to extend the nucleotide sequence of the original template during creation of the engineered strand of nucleic acid.
- the engineered strand of nucleic acid is created from the original template by a series of manipulations that result in the presence of a predetermined sequence at one end thereof and a hairpin structure. It is these two features that allow amplification using only a single primer.
- any nucleic acid in purified or nonpurified form, can be utilized as the starting material for the processes described herein provided it contains or is suspected of containing the target nucleic acid sequence to be amplified.
- the starting material employed in the process may be, for example, DNA or RNA, including messenger RNA, which DNA or RNA may be single stranded or double stranded.
- a DNA-RNA hybrid which contains one strand of each may be utilized.
- a mixture of any of these nucleic acids may also be employed, or the nucleic acids produced from a previous amplification reaction herein using the same or different primers may be utilized.
- the target nucleic acid sequence to be amplified may be a fraction of a larger molecule or can be present initially as a discrete molecule.
- the starting nucleic acid may contain more than one desired target nucleic acid sequence which may be the same or different. Therefore, the present process may be useful not only for producing large amounts of one target nucleic acid sequence, but also for amplifying simultaneously more than one different target nucleic acid sequence located on the same or different nucleic acid molecules.
- the nucleic acids may be obtained from any source, for example: genomic or cDNA libraries, plasmids, cloned DNA or RNA, or from natural DNA or RNA from any source, including bacteria, yeast, viruses, and higher organisms such as plants or animals.
- the nucleic acid can be naturally occurring or synthetic, either totally or in part. Techniques for obtaining and producing the nucleic acids used in the present processes are well known to those skilled in the art. If the nucleic acid contains two strands, it is necessary to separate the strands of the nucleic acid before it can be used as the original template, either as a separate step or simultaneously with the synthesis of the primer extension products. Additionally, if the starting material is first strand DNA, second strand DNA may advantageously be created by processes within the purview of those skilled in the art and used as the original template from which the engineered template is created.
- First strand cDNA and mRNA are particularly useful as the original template for the present methods. Suitable methods for generating DNA templates are known to and readily selected by those skilled in the art.
- 1 st strand cDNA is synthesized in a reaction where reverse transcriptase catalyzes the synthesis of DNA complementary to any RNA starting material in the presence of an oligodeoxynucleotide primer and the four deoxynucleoside triphosphates, dATP, dGTP, dCTP, and TTP.
- the reaction is initiated by the non-covalent bonding of the oligo-deoxynucleotide primer to the 3′ end of mRNA followed by stepwise addition of the appropriate deoxynucleotides as determined by base pairing relationships with the mRNA nucleotide sequence, to the 3′ end of the growing chain.
- all mRNA in a sample can be used to generate first strand cDNA through the annealing of oligo dT to the poly A tail of the mRNA.
- a primer 20 and a boundary oligonucleotide 30 are annealed to the original template 10 .
- a strand of nucleic acid complementary to the portion of the original template beginning at the 3′ end of the primer up to about the 5′ end of the boundary oligonucleotide is polymerized.
- the primer 20 that is annealed to the original template includes a portion 25 that anneals to the original template and optionally a portion 22 of predetermined sequence that preferably does not anneal to the template, and optionally a restriction site 23 between portions 22 and 25 .
- the primer can be any gene-specific internal sequence of the mRNA or oligo dT which will anneal to the unique poly A tail of the mRNA.
- the primer anneals to the original template adjacent to the target sequence 12 to be amplified. It is contemplated that the primer can anneal to the original template upstream of the target sequence (or downstream in the case, e.g., of an mRNA original template) to be amplified, or that the primer may overlap the beginning of the target sequence 12 to be amplified as shown in FIG. 1.
- the predetermined sequence of portion 22 of the primer is selected so as to provide a sequence to which the single primer used during the amplification process can hybridize as described in detail below.
- the predetermined sequence is not native in the original template and does not anneal to the original template, as shown in FIG. 1.
- the predetermined sequence may include a restriction site useful for insertion of a portion of the engineered template into an expression vector as described more fully hereinbelow.
- the boundary oligonucleotide 30 that is annealed to the original template serves to terminate polymerization of the nucleic acid.
- Any oligonucleotide capable of terminating nucleic acid polymerization may be utilized as the boundary oligonucleotide 30 .
- the boundary oligonucleotide includes a first portion 35 that anneals to the original template 10 and a second portion 32 that is not susceptible to an extension reaction. Techniques to prevent the boundary oligo from acting as a site for extension are within the purview of one skilled in the art.
- portion 32 of the boundary oligo 30 may be designed so that it does not anneal to the original template 10 as shown in FIG. 1.
- the boundary oligonucleotide 30 prevents further polymerization but does not serve as a primer for nucleic acid synthesis because the 3′ end thereof does not hybridize with the original template 10 .
- the 3′ end of the boundary oligo 30 might be designed to include locked nucleic acid to achieve the same effect. Locked nucleic acid is disclosed for example in WO 99/14226, the contents of which are incorporated herein by reference. Those skilled in the art will envision other ways of ensuring that no extension of the 3′ end of the boundary oligo occurs.
- Oligonucleotides described herein may be synthesized using established methods for oligonucleotide synthesis which are well known in the art.
- Oligonucleotides, including primers of the present invention include linear oligomers of natural or modified monomers or linkages, such as deoxyribonucleotides, ribonucleotides, and the like, which are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to monomer interactions such as Watson-Crick base pairing.
- a primer is typically single-stranded, but may be double-stranded.
- Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers known in the art may be useful for the methods of the present disclosure.
- a primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers may be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
- Polymerization of nucleic acid can be achieved using methods known to those skilled in the art. Polymerization is generally achieved enzymatically, using a DNA polymerase or reverse transcriptase which sequentially adds free nucleotides according to the instructions of the template. The selection of a suitable enzyme to achieve polymerization for a given template and primer is within the purview of those skilled in the art. In certain embodiments, the criteria for selection of polymerases includes lack exonuclease activity or DNA polymerases which do not possess a strong exonuclease activity. DNA polymerases with low exonuclease activity for use in the present process may be isolated from natural sources or produced through recombinant DNA techniques.
- polymerases that may be used, are, without limitation, T7 Sequenase v. 2.0, the Klenow Fragment of DNA polymerase I lacking exonuclease activity, the Klenow Fragment of Taq Polymerase, exo.-Pfu DNA polymerase, Vent. (exo.-) DNA polymerase, and Deep Vent. (exo-) DNA polymerase.
- a restriction oligonucleotide 70 is annealed to the starting material 100 at a preselected location.
- the restriction oligonucleotide provides a double stranded portion on the starting material containing a restriction site 72 .
- Suitable restriction sites include, but are not limited to Xho I, Spe I, Nhe1, Hind III, Nco I, Xma I, Bg1 II, Bst I, and Pvu I.
- the starting material Upon exposure to a suitable restriction enzyme, the starting material is digested and thereby shortened to remove unnecessary sequence while preserving the desired target sequence 12 (or portion thereof) to be amplified on what will be used as the original template 110 .
- a primer 20 is annealed to the original template 110 (see FIG. 2B) adjacent to or overlapping with the target sequence 12 as described above in connection with previous embodiments.
- a strand of nucleic acid 40 complementary to the portion of the original template between the 3′ end of the primer 20 and the 5′ end of the original template 110 is polymerized.
- the newly synthesized complementary strand is separated from the original template by any suitable denaturing method including physical, chemical or enzymatic means.
- Strand separation may also be induced by an enzyme from the class of enzymes known as helicases or the enzyme RecA, which has helicase activity and in the presence of riboATP is known to denature DNA.
- the reaction conditions suitable for separating the strands of nucleic acids with helicases are described by Cold Spring Harbor Symposia on Quantitative Biology, Vol.
- the newly synthesized complementary strand thus includes sequences provided by the primer 20 (e. g., the predetermined sequence 22 , the optional restriction site 23 and the annealing portion 25 of the primer) as well as the newly synthesized portion 45 that is complementary to the portion of the original template 10 between the location at which the primer 20 was annealed to the original template 10 and either the portion of the original template 10 to which the boundary oligonucleotide 30 was annealed or through to the shortened 5′ end of the original template. See FIG. 3.
- multiple rounds of polymerization using the original template and a primer are performed to produce multiple copies of the newly synthesized complementary strand for use in subsequent steps. It is contemplated that 2 to 10 rounds or more (preferably, 15-25 rounds) of linear amplification can be performed when a DNA template is used. Making multiple copies of the newly synthesized complementary strand at this point in the process (instead of waiting until the entire engineered template is produced before amplifying) helps ensure that accurate copies of the target sequence are incorporated into the engineered templates ultimately produced. It is believed that multiple rounds of polymerization based on the original template provides a greater likelihood that a better representation of all members of the library will be achieved, therefore providing greater diversity compared to a single round of polymerization.
- the next step in preparing the engineered template involves annealing a nested oligonucleotide 50 to the 3′ end of the newly synthesized complementary strand, for example as shown in FIG. 3.
- the nested oligonucleotide 50 provides a template for further polymerization necessary to complete the engineered template.
- Nested oligonucleotide 50 includes a portion 52 that does not hybridize and/or includes modified bases to the newly synthesized complementary strand, thereby preventing the nested oligonucleotide from serving as a primer.
- Nested oligonucleotide 50 also includes a portion 55 that hybridizes to the 3′ end of the newly synthesized complementary strand.
- Nested oligonucleotide 50 may optionally also define a restriction site 56 .
- the final portion 58 of nested oligonucleotide 50 contains a hairpin structure. From the point at which portion 55 extends beyond the 3′ end of the beginning the newly synthesized complementary strand, the nested oligonucleotide serves as a template for further polymerization to form the engineered template. It should be understood that the nested oligo may contain part of the target sequence (if part thereof was truncated in forming the original template) or may include genes that encode a polypeptide or protein (or portion thereof) such as, for example, one or more CDR's or Framework regions or constant regions of an antibody.
- polymerization will extend the newly synthesized complementary strand by adding additional nucleic acid 60 that is complementary to the nested oligonucleotide as shown in FIG. 3.
- an enzyme lacking exonuclease activity may be employed to prevent the 3′ end of the nested oligo from acting as a primer.
- the engineered template 120 is separated from the nested oligonucleotide 50 by techniques well known to those skilled in the art such as, for example, heat denaturation.
- the resulting engineered template 120 contains a portion derived from the original primer 20 , portion 45 that is complementary to a portion of the original template, and portion 65 that is complementary to a portion of the nested oligonucleotide and includes a hairpin structure 68 , and a portion 69 that is complementary to portion 45 .
- the 3′ end of engineered template 120 includes a portion containing a sequence that is complementary to primer 20 .
- the 3′ end of engineered template 120 includes portion 22 ′ containing a sequence that is complementary to the predetermined sequence of portion 22 of primer 20 .
- portion 22 ′ containing a sequence that is complementary to the predetermined sequence of portion 22 of primer 20 . This allows for amplification of the desired sequence contained within engineered template 120 using a single primer having the same sequence as the predetermined sequence of primer portion 22 (or a primer that is complementary thereto) using techniques known to those of ordinary skill in the art.
- the 3′ end of engineered template 120 includes poly A portion that is complementary to the oligo dT primer.
- any sequence along portion 45 can be selected for use as the primer to be annealed to portion 69 once the engineered template is denatured for single primer amplification.
- the primer may include a non-annealing portion, such as, for example, a portion defining a restriction site.
- hairpin structure 68 there is internal self annealing between the 5′ end predetermined sequence and the 3′ end sequence which is complementary to the predetermined sequence on the engineered template. Upon denaturation and addition of a primer having the predetermined sequence, the primer will hybridize to the template and amplification can proceed.
- the products may be detected using any of the techniques known to those skilled in the art.
- methods used to detect nucleic acids include, without limitation, hybridization with allele specific oligonucleotides, restriction endonuclease cleavage, single-stranded conformational polymorphism (SSCP), analysis gel electrophoresis, ethidium bromide staining, fluorescence resonance energy transfer, hairpin FRET essay, and TaqMan assay.
- restriction sites 23 and 66 or any internal restriction site can be used to digest the strand so that the target nucleic acid sequence can be ligated into a suitable expression vector.
- the vector may then be used to transform an appropriate host organism using standard methods to produce the polypeptide or protein encoded by the target sequence.
- the methods described herein are used to amplify target sequences encoding antibodies or portions thereof, such as, for example the variable regions (either light or heavy chain) using cDNA of an antibody.
- a library of antibodies can be amplified and screened.
- first strand cDNA can be produced and digested to provide an original template.
- a primer can be designed to anneal upstream to a selected complementary determining region (CDR) so that the newly synthesized nucleic acid strand includes the CDR.
- CDR complementary determining region
- the primer may be designed to anneal to the heavy chain framework one (FR1) region.
- First strand cDNA to be used as the original template was generated from 2 ⁇ g of human peripheral blood lymphocyte (PBL) mRNA with an oligo-dT primer using the SuperScript II First Strand Synthesis Kit (Invitrogen) according to the manufacturer's instructions.
- the 1 st strand cDNA product was purified over a QIAquick spin column (QIAGEN PCR Purification Kit) and eluted in 400 ⁇ L of nuclease-free water.
- the second strand cDNA reaction contained 5 ⁇ L of 1 st strand cDNA original template, 0.5 ⁇ M primer JMX26VK1a, 0.5 ⁇ M blocking oligo CKLNA1, 0.2 mM dNTPs, 5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems), 1 ⁇ GeneAmp Gold Buffer (15 Mm Tris-HCl, pH 8.0, 50 mM KCl), and 1.5 mM MgCl 2 . The final volume of the reaction was 98 ⁇ L.
- the sequence of primer JMX26VK1a which hybridizes to the framework 1 region of VK1a genes, was 5′ GTC ACT CAC GAA CTC ACG ACT CAC GGA GAG CTC RAC ATC CAG ATG ACC CAG 3′ (Seq. ID No. 1) where R is an equal mixture of A and G.
- LNA locked nucleic acid
- JK14TSHP a nested/hairpin oligo designated “JK14TSHP” was added to give a final concentration of 20 ⁇ M.
- the sequence of JK14TSHP was 5′ CCT TAG AGT CAC GCT AGC GAT TGA TTG ATT GAT TGATTG TTT GTG ACT CTA AGG TTG GCG CGC CTT CGT TTG ATY TCC ACC TTG GTC C(ps)T(ps)G(ps)P 3′ (Seq. ID No. 3) where Y is an equal mixture of C and T and (ps) are phosphorothioate backbone linkages and P is a 3′ propyl group.
- oligo extension reaction For nested oligo extension reaction, two cycles of 94° C. for 1 minute, 56° C. for 15 seconds, and 72° C. for 1 minute were performed, followed by a 10 minute incubation at 72° C. to allow complete extension of the hairpin.
- the reaction products were purified over a QIAquick spin column (QIAgen PCR Purification Kit) and eluted in 50 ⁇ L of nuclease-free water.
- the efficiency of the nested oligo extension reaction was determined by amplifying the products with either a primer set specific for the engineered product or a primer set that detects all VK1a/JK14 second strand cDNA products (including the engineered product). For specific detection of engineered product, a 10 ⁇ L aliquot was amplified for 20 or 25 cycles with primers designated “JMX26” and “TSDP”. Primer JMX26 hybridizes to the 5′ end of JMX26VK1a, the framework 1 primer used in the second strand cDNA reaction. Primer TSDP hybridizes to the hairpin-loop sequence added to the 3′ ends of the second strand cDNAs in the nested oligo extension reaction.
- primer JMX26 was 5′ GTC ACT CAC GAA CTC ACG ACT CAC GG 3′ (Seq. ID No. 4).
- sequence of primer TSDP was 5′ CAC GCT AGC GAT TGA TTG ATT G 3′ (Seq. ID No. 5).
- a 10 ⁇ L aliquot was amplified for 20 or 25 cycles with primers JMX26 and JK14.
- primer JK14 which hybridizes to the framework 4 region of JK1 and JK4 genes, was 5′ GAG GAG GAG GAG GAG GAG GAG GGC GCG CCT GAT YTC CAC CTT GGT CCC 3′ (Seq. ID No. 6). Both reactions contained 1 ⁇ GeneAmp Gold Buffer, 1.5 mM MgCl 2 , 7.5% glycerol, 0.2 mM dNTPs, and 0.5 ⁇ M of each primer in a final volume of 50 ⁇ L.
- Conditions that were previously shown to amplify a 352 bp stem-15 bp loop DNA product were as follows: 10 pg of the stem-loop DNA, 2 ⁇ M primer, 50 mM Tris-HCl, pH 9.0, 1.5 mM MgCl 2 , 15 mM (NH 4 ) 2 SO 4 , 0.1% Triton X-100, 1.7 M betaine, 0.2 mM dNTPs, and 2.5 units of Z-Taq DNA Polymerase (Takara Shuzo) in a final volume of 50 ⁇ L.
- the thermal cycling conditions were an initial denaturation step of 96° C. for 2.5 minutes, 35 cycles of 96° C. for 30 seconds, 64° C.
- Oligonucleotides containing the modified bases 5-methyl-2′-deoxycytidine and/or 2-amino-2′-deoxyadenosine have been shown to prime much more efficiently than unmodified oligonucleotides at primer binding sites located within hairpin structures (Lebedev et al. 1996. Genetic Analysis: Biomolecular Engineering 13, 15-21). These modifications work by increasing the melting temperature of the primer, allowing the annealing step of the amplification to be performed at a higher temperature. JMX26 primers containing ten 5-methyl-2′-deoxycytidines or seven 2-amino -2′-deoxyadenosines have been synthesized.
- Amplified fragments are cloned by Sac I/Asc I into an appropriate expression vector that contains, in frame, the remaining portion of the kappa constant region.
- Suitable vectors include pRL5 and pRL4 vectors (described in U.S. Provisional Application 60/254,411, the disclosure of which is incorporated hererin by reference), fdtetDOG, PHEN1, and pCANTAB5E. Individual kappa clones can be sequenced.
- VK repertoire Further coverage of the VK repertoire is achieved by using the above protocols with a panel of primers for the generation of the second strand DNA.
- the primers contain JMX26 sequence, a Sac I restriction site, and a region that anneals to 1 st strand cDNA in the framework 1 region of human antibody kappa light chain genes.
- the antibody annealing sequences were derived from the VBase database primers (www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html) which were designed based on the known sequences of human antibodies and are reported to cover the entire human antibody repertoire of kappa light chain genes.
- JMX26Vk1a GTCACTCACGAACTCACGACTCACGGA GAGCTC RACATCCAGATGACCCAG (Seq. ID No.7)
- JMX26Vk1b GTCACTCACGAACTCACGACTCACGGA GAGCTC GMCATCCAGTTGACCCAG (Seq. ID No.8)
- JMX26Vk1c GTCACTCACGAACTCACGACTCACGGA GAGCTC GCCATCCRGATGACCCAG (Seq. ID No.9)
- JMX2GVk1d GTCACTCACGAACTCACGACTCACGGA GAGCTC GTCATCTGGATGACCCAG (Seq.
- JMX26Vk2a JMX26Vk2a GTCACTCACGAACTCACGACTCACGGA GAGCTC GATATTGTGATGACCCAG (Seq. ID No.11) JMX26Vk2b GTCACTCACGAACTCACGACTCACGGA GAGCTC GATRTTGTGATGACTCAG (Seq. ID No.12) JMX26Vk3a GTCACTCACGAACTCACGACTCACGGA GAGCTC GAAATTGTGTTGACRCAG (Seq. ID No.13) JMX26Vk3b GTCACTCACGAACTCACGACTCACGGA GACCTC GAAATAGTGATGACGCAG (Seq.
- JMX2GVk3c GTCACTCACGAACTCACGACTCACGGA GAGCTC GAAATTGTAATGACACAG (Seq. ID No.15) JMX26Vk4a GTCACTCACGAACTCACGACTCACGGA GAGCTC GACATCGTGATGACCCAG (Seq. ID No.16) JMX26Vk5a GTCACTCACGAACTCACGACTCACGGA GAGCTC GAAACGACACTCACGCAG (Seq. ID No.17) JMX2GVk6a GTCACTCACGAACTCACGACTCACGGA GAGCTC GAAATTGTGCTGACTCAG (Seq. ID No.18) JMX2Gvk6b GTCACTCACGAACTCACGACTCACGGA GAGCTC GATGTTGTGATGACACAG (Seq. ID No.19)
- R is an equal mixture of A and G
- M is an equal mixture of A and C
- Y is an equal mixture of C and T
- W is an equal mixture of A and T
- S is an equal mixture of C and G.
- JMX26 Similar protocols are applied to the amplification of both heavy chain and lambda light chain genes. JMX26, or another primer without antibody specific sequences, is used for each of those applications. If JMX26 is used, the second strand DNA is generated with the primers listed below which contain JMX26 sequence, a restriction site (Sac I for lambda, Xho I for heavy chains), and a region that anneals to 1 st strand cDNA in the framework 1 region of human antibody lambda light chain or heavy chain genes.
- the antibody annealing sequences were derived from the VBase database primers (www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html) which were designed based on the known sequences of human antibodies and are reported to cover the entire human antibody repertoire of lambda light chain and heavy chain genes.
- Lambda Light Chain Framework 1 Specific Primers JMX2GVL1a GTCACTCACGAACTGACGACTCACGGA GAGCTC CAGTCTGTGCTGACTCAG (Seq. ID No. 20) JMX2GVL1b GTCACTCACGAACTCACGACTCACGGA GAGCTC CAGTCTGTGYTGACGCAG (Seq. ID No. 21) JMX264VL1C CTCACTCACGAACTCACGACTCACGGA GAGCTC CAGTCTGTCGTGACGCAG (Seq. ID No. 22) JMX26VL2 GTCACTCACGAACTCACGACTCACGGA GAGCTC CAGTCTGCCCTGACTCAG (Seq. ID No.
- JMX26VL3a GTCACTCACGAACTCACCACTCACGGA GAGCTC TCCTATGWGCTGACTCAG
- JMX26VL3b GTCACTCACGAACTCACGACTCACGGA GAGCTC TCCTATGAGCTGACACAG
- JMX26VL3c GTCACTCACGAACTCACGACTCACGGA GAGCTC TCTTCTGAGCTGACTCAG
- JMX26VL3d GTCACTCACGAACTCACGACTCACGGA GAGCTC TCCTATGAGCTGATGCAG (Seq. ID No.
- R is an equal mixture of A and G
- M is an equal mixture of A and C
- Y is an equal mixture of C and T
- W is an equal mixture of A and T
- S is an equal mixture of C and G.
- Heavy Chain Framework 1 Specific Primers: JMX24VH1a GTCACTCACGAACTCACGACTCACGGA ctcgag CAGGTKCAGCTGGTGCAG (Seq. ID No. 35) JMX24VH1b GTCACTCACGAACTCACGACTCACGGA ctcgag CAGGTCCAGCTTGTGCAG (Seq. ID No. 36) JMX26VH1c GTCACTCACGAACTCACGACTCACGGA ctcgag SAGGTCCAGCTGGTACAG (Seq. ID No.
- JMX26VH3b GTCACTCACCAACTCACGACTCACGCA ctcgag CAGGTGCAGCTGGTGGAG (Seq. ID No. 42) JMX26VH3c CTCACTCACGAACTCACGACTCACGGA ctcgag GAGGTGCAGCTGTTGGAG (Seq. ID No. 43) JMX26VH4a GTCACTCACGAACTCACGACTCACGGA ctcgag CAGCTGCAGCTGCAGGAG (Seq. ID No. 44) JMX26VH4b GTCACTCACGAACTCACCACTCACGGA ctcgag CAGGTGCAGCTACAGCAG (Seq. ID No.
- R is an equal mixture of A and G
- K is an equal mixture of G and T
- S is an equal mixture of C and G.
- Blocking oligos for the constant domain of IgM, IgG, and lambda chains are designed. Essentially a region downstream of that required for cloning the genes is identified, and within that region, a sequence useful for annealing a blocking oligo is determined. For example with IgG heavy chains, a native Apa I restriction site present in the CH1 domain can be used for cloning. Generally, the boundary oligo is located downstream of that native restriction site. Compatible nested oligos are then designed which contained all the elements described previously.
- the lambda light chain genes are cloned as is described above for the kappa light chain genes.
- amplified IgG heavy chain fragments are cloned by Xho I/Apa I into an appropriate expression vector that contains, in frame, the remaining portion of the CH1 constant region. Suitable vectors include pRL5, pRL4, fdtetDOG, PHEN1,and pCANTAB5E.
- Amplified IgM heavy chain fragments are cloned by Xho I/EcoR I into an appropriate expression vector that contains, in frame, the remaining portion of the CH1 constant region.
- the EcoR I site is native to the IgM CH1 domain. Libraries co-expressing both light chains and heavy chains can be screened or selected for Fabs with the desired binding activity.
- Human peripheral blood lymphocyte (PBL) mRNA was used as the original template to generate the first strand cDNA with ThermoScript RT-PCR System (Invitrogen Life Technologies).
- a phosphoramidate oligonucleotide (synthesized by Annovis Inc. Aston, Pa.) was also included in the reverse transcription reaction.
- the phosphoramidate oligonucleotide serves as a boundary for reverse transcriptase.
- the first strand cDNA synthesis was terminated at the location where the phosphoramidate oligonucleotide anneals with the mRNA.
- the phosphoramidate oligonucleotide, PN-1 was designed to anneal with the framework I region of immunoglobulin (Ig) heavy chain VH3 genes and PN-VH5 was designed to anneal with the framework I region of all the Ig heavy chain genes.
- a control for first strand cDNA synthesis was also set up by not including the phosphoramidate blocking oligonucleotide.
- the first strand cDNA product was purified by QIAquick PCR Purification Kit (QIAGEN).
- Phosphoramidate Framework I Blocking Oligonucleotides for Ig Heavy Chain Genes have the following sequences: (Seq. ID No. 49) PN-1 5′ GCCTCCCCCAGACTC 3′ (Seq. ID No. 50) PN-VH5 5′ GCTCCAGACTGCACCAGCTGCAC(C/T)TCGG 3′
- the blocking efficiency in first strand cDNA synthesis was examined by PCR reactions using blocking check primers and primer CM1, dNTPs, Advantage-2 DNA polymerase mix (Clontech), the reaction buffer, and the first strand cDNA synthesis product.
- PCR was performed on a PTC-200 thermal cycler (MJ Research) by heating to 94° C. for 30 seconds and followed by cycles of 94° C. for 15 second, 60° C. for 15 second, and 72° C. for one minute.
- the blocking check primers were designed to anneal with the leader sequences of IgM heavy chain genes.
- CM1 which hybridizes with the CH1 region of IgM, was 5′ GCTCAC ACTAGT AGGCAGCTCAGCAATCAC 3′ (Seq. ID No. 51). Blocking was analyzed by gel electrophoresis of the PCR products. With appropriate number of cycles, less PCR product was observed from the reverse transcription reactions containing the blocking oligonucleotides than the one does not contain the blocking oligonucleotides, an indication that termination of first strand cDNA synthesis was provided by the hybridization of the blocking oligonucleotides.
- sequences of the blocking check Primers for Ig heavy chain genes have the following sequences: H1/7blck 5′ C TGG ACC TGG AGG ATC C 3′ (Seq. ID No. 52) H1blck2 5′ C TGG ACC TGG AGG GTC T 3′ (Seq. ID No. 53) H1blck3 5′ C TGG ATT TGG AGG ATC C 3′ (Seq. ID No. 54) H2blck1 5′ GACACACTTTGCTCCACG 3′ (Seq. ID No. 55) H2blck2 5′ GAC ACA CTT TGC TAC ACA 3′ (Seq. ID No.
- H3blck 5′ TGGGGCTGAGCTGGGTTT 3′ (Seq. ID No. 57) H3blck2 5′ TG GGA CTG AGC TGG ATT T 3′ (Seq. ID No. 58) H3blck3 5′ TT GGG CTG AGC TGG ATT T 3′ (Seq. ID No. 59) H3blck4 5′ TG GGG CTC CCC TGG GTT T 3′ (Seq. ID No. 60) H3blck5 5′ TT GGG CTG AGC TGG CTT T 3′ (Seq. ID No.
- H3blck6 5′ TT GGA CTG AGC TGG GTT T 3′
- H3blck7 5′ TT TGG CTG AGC TGG GTT T 3′
- H4blck 5′ AAACACCTGTGGTTCTTC 3′
- H4blck2 5′ AAG CAC CTG TGG TTT TTC 3′
- H5blck 5′ GCGTCAACCGCCATCCT 3′ Seq. ID No. 66
- H6blck 5′ TCTGTCTCCTTCCTCATC 3′ (Seq. ID No. 67)
- the purified first strand cDNA synthesis product was used in a nested oligo extension reaction with a hairpin-containing nesting oligonucleotide, dNTPs, Advantage-2, DNA polymerase mix (Clontech), and the reaction buffer.
- the extension reaction was performed with a GeneAmp PCR System 9700 thermocyler (PE Applied Biosystems). It was heated to 94° C. for 30 seconds and followed by ten cycles of 94° C. for 15 seconds, appropriate annealing temperature for each nesting oligonucleotide for 15 seconds, ramping the temperature to 90° C. at 10% of the normal ramping rate, and 90° C. for 30 seconds.
- the resulted heavy chain gene should contain a hairpin structure.
- GGAGCAGAGGTGAAAAAGCCCGGGGAAAA 3′ Nesting Oligonucleotides for Ig VH6 Heavy Chain Genes: hpVH6 5′ +E,usn CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAGGTA GAG CTG GAG GAG TCA (Seq. ID No. 91)
- GGT CCA GGA CTG GTG AAG CCC AAA 3′ Nesting Oligonucleotides for Ig VH7 Heavy Chain Genes: hpVH7 5′ CTCGAG GGCCCGCGAAAGCGGGCCCTCGAG GAG GTG GAG CTG GTG CAA TCT (Seq. ID No.
- Products from the nesting oligo extension reaction i.e. the engineered template
- the amplification reaction was performed with a GeneAmp PCR System 9700 thermocyler (PE Applied Biosystems). It was initially heated to 94° C. for 30 seconds and followed by thirty to forty cycles of 94° C. for 15 seconds, appropriate annealing temperature for 15 seconds, ramping the temperature to 90° C. at 10% of the normal ramping speed, and at 90° C. for 30 seconds.
- the amplified product was examined by electrophoresis to be of the expected size, ⁇ 0.7 kb.
- the amplified fragments were cloned into an expression vector and their sequences were confirmed to be human IgM.
- the same protocol as example 3 is employed using mRNA of PBL from a human donor immunized with hepatitis B surface antigen and the phosphoramidate boundary oligonucleotides designed to anneal with the leader sequence of the Ig heavy chain genes.
- the phosphoramidate leader boundary oligonucleotides for Ig heavy chain genes have the following sequences: PNVH31d 5′ CACCTCACACTGGACACCTTT 3′ (Seq. ID No. 95) PNVH41d 5′ CTGGGACAGGACCCATCTGGG 3′ (Seq. ID No. 96) PNVHL1d 5′ TGGGAGTGGGCACCTGTGG 3′ (Seq. ID No.
- the blocking efficiency in first strand cDNA synthesis is examined by PCR reactions using dNTPs, Advantage-2 DNA polymerase mix (Clontech), the reaction buffer, the first strand cDNA synthesis product, the blocking check primers in Example 3, and the pooled primer mixture of CG1Z, CG2speI, CG3speI, and CG4SpeI.
- the sequence of primer CG1Z, which hybridized with the CH1 region of IgG1, is 5′GCATGTACTAGTTTTGTCACAAGATTTGGG 3′.
- primer CG2speI which hybridized with the CH1 region of IgG2, is 5′AAGGAAACTAGTTTTGCGCTCAACTGTCTTGTCCACCTT 3′.
- sequence of primer CG3speI which hybridized with the CH1 region of IgG3, is 5′AAGGAAACTAGTGTCACCAAGTGGGGTTTTGAGCTC 3′.
- sequence of primer CG4speI which hybridized with the CH1 region of IgG4, is 5′AAGGAAACTAGTACCATATTTGGACTCAACTCTCTTG 3′.
- PCR is performed on a PTC-200 thermal cycler (MJ Research) by heating to 94° C. for 30 seconds before the following cycle is run, 94° C. for 15 second, 60° C. for 15 second, and 72° C. for one minute.
- the PCR products were analyzed by gel electrophoresis. With appropriate number of cycles less PCR products were observed from reverse transcription reactions containing the blocking oligonucleotide than the one does not contain blocking oligonucleotide, an indication that termination of first strand cDNA synthesis was provided by hybridization of the leader boundary oligonucleotides.
- Example 3 The same protocol as Example 3 is employed with nesting oligonucleotides having the following sequences are used.
- Example 3 The sample protocol as Example 3 was employed using CG1Z, CG2speI, CG3speI, or CG4SpeI as the primer.
- the amplified IgG heavy chain fd hairpin fragments are analyzed by gel electrophoresis.
- the ⁇ 0.7 kb fragment is separated from the primers by cutting out the gel slice and the DNA was collected by electroelution.
- the eluted DNA was precipitated by ethanol and resuspended in water. It is digested with restriction enzymes XhoI and SpeI and purified by the QIAquick PCR Purification Kit (QIAGEN).
- the purified XhoI-SpeI fragment is ligated into a suitable plasmid into which the light chain kappa genes amplified from the same donor had previously been cloned.
- the ligated reaction was transformed into E.
- the XL-1 Blue cells electroporated with the ligation reaction of the phagemid vector and the heavy chain Fd fragments were grown in SOC medium at 37° C. with shaking for one hour.
- SOC medium is 20 mM glucose in SB medium which contains 1% MOPS hemisodium salt, 3% Bacto Tryptone, and 2% Bacto Yeast Extract.
- Cells transformed with the plasmid were selected by adding carbenicillin to the culture and they were grown for two hours before infected with a helper phage, VCSM13. After two hours XL-1 Blue cells infected with the helper phage were selected by adding Kanamycin to the culture and the infected cells were amplified overnight by growing at 37° C. with shaking.
- the amplified phages were harvested by precipitating with polyethylene glycol (PEG) from the culture supernatant.
- PEG precipitated phages were collected by centrifugation. They were resuspended in 1% bovine serum albumin (BSA) in TBS buffer and used in panning for selecting human IgG antibodies that bind with the hepatitis B surface antigen.
- BSA bovine serum albumin
- the resuspended phages were bound with the hepatitis B surface antigen immobilized on the ELISA plate (Costar).
- the unbound phages were washed off with a washing buffer (0.5% Tween 20 in PBS) and the bound phages were eluted off the plate with a phage elution buffer (0.1M HCl/glycine, pH 2.2, 1 mg/ml BSA) and neutralized with a neutralization buffer (2M Tris Base).
- the eluted phages were infected with E. coli ER strain ⁇ F′ proA + B + lac1 q ⁇ (lacZ) M15/fhuA2 (ton A) ⁇ (lac-proAB) supE thi-1 ⁇ (hsdMS-mcrB)5 ⁇ , followed by infection with VCSM13 helper phage.
- the panning procedure for selecting antibodies bound to hepatitis B surface antigen were repeated three more times.
- Phages eluted at the fourth round of panning were infected with E. coli Top10F′ strain ⁇ F′ lac1 q , Tn10 (Tet R mcrA ⁇ (mrr-hsdRMS-mcrBC) ⁇ 8(lacZ ⁇ m15 ⁇ lacx74 deoR recA1 araD13 ⁇ (ara-leu)7697 galU galK [sL(Str R ) endA1 nupG) and plated on LB-agar plates containing carbenicilin and tetracycline. Individual clones were picked from the plates and grown overnight in SB medium containing carbenicilin and tetracycline.
- the IgG Fab fragment will be secreted into the culture supernatant.
- the next morning cells were removed from these cultures by centrifugation and the culture supernatant was screened in ELISA assay for binding to hepatitis B surface antigen immobilized on the ELISA plates. To reduce false positives the ELISA plates were pre-blocked with BSA before binding with the Fab fragments in culture supernatant. The non-binding Fab fragments were washed off by a washing solution (0.05% Tween 20 in PBS).
- the IgG heavy chain genes of positive clones from ELISA screening were characterized by DNA sequencing. Plasmid DNA was extracted from the positive clones and sequenced using primers leadVHpAX, NdP, or SeqGZ (Retrogen, San Diego, Calif.). The sequencing primers have the following sequences: VBVH3A 5′ GAGCCGCACGAGCCCCTCGAGGARGTGCAGCTGGTGGAG 3′ (Seq. ID No. 122) VBVH 3B 5′ GAGCCGCACGAGCCCCTCGAGGAGGTGCAGCTGGTGGAG 3′ (Seq. ID No.
- VBVH 3C 5′ GAGCCGCACGAGCCCCTCGAGGAGGTGCAGCTGTTGGAG 3′ (Seq. ID No. 124)
- VBVH 4A 5′ GAGCCGCACGAGCCCCTCGAGCAG(CG)TGCAGCTGCAGGAG 3′ (Seq. ID No. 125)
- VBVH 4B 5′ GAGCCGCACGAGCCCCTCGAGCAGGTGCAGCTACAGCAG 3′ (Seq. ID No. 126)
- LeadVHPAX 5′ GCGGCGCAGCCGGCGATGGCG 3′ (Seq. ID No. 127)
- NdP 5′ AGCGTAGTCCGGAACGTCGTACGG (Seq. ID No. 128) SeqGZ 5′ GAAGTAGTCCTTGACCAG 3′ (Seq. ID No. 129)
- the same protocol as example 3 is employed using the phosphoramidate boundary oligonucleotides designed to hybridize with the leader sequence of the kappa light chain genes.
- the phosphoramidate leader boundary oligonucleotides for kappa light chain genes have the following sequences: (Seq. ID No. 130) PNK11d: 5′ T GTC ACA TCT GGC ACC TGG 3′ (Seq. ID No. 131) PNK21d: 5′ TC CCC ACT GGA TCC AGG GAC 3′ (Seq. ID No. 132) PNK31d: 5′ C TCC GGT GGT ATC TGG GAG 3′ (Seq. ID No.
- PNK41d 5′ TC CCC GTA GGC ACC AGA GA 3′ (Seq. ID No. 134)
- PNK51d 5′ TC TGC CCT GGT AT C AGA GAT 3′ (Seq. ID No. 135)
- PNK61d 5′ C ACC CCT GGA GGC TGG AAC 3′
- the blocking efficiency in first Strand cDNA Synthesis was examined by PCR reactions using blocking check primers and primer CK1DX2, dNTPs, Advantage-2 DNA polymerase mix (Clontech), the reaction buffer, and the first strand cDNA synthesis product.
- PCR was performed on a PTC-200 thermal cycler (MJ Research) by heating to 94° C. for 30 seconds and followed by cycles of 94° C. for 15 second, 60° C. for 15 second, and 72° C. for one minute.
- the blocking check primers were designed to anneal with the leader sequences of kappa light chain genes.
- CK1DX2 which hybridizes with the constant region of Kappa light chain, was 5′AGACAGTGAGCGCCGTCTAGAATTAACACTCTCCCCTGTTGAAGCTCTTTGTGACGGGCGAACTCAG 3′.
- Blocking was analyzed by gel electrophoresis of the PCR products. With appropriate number of cycles less PCR products was observed from reverse transcription reactions containing the blocking oligonucleotide than one that does not contain blocking oligonucleotide, an indication that termination of first strand cDNA synthesis was provided by hybridization of the leader boundary oligonucleotides.
- Blocking Check Primers for Kappa Light Chain Genes have the following sequences: K1b1ck: 5′ CTCCGAGGTGCCAGATGT 3′ (Seq. ID No. 137) K1/2b1ck2: 5′ GCT CAG CTC CTG GGG CT 3′ (Seq. ID No. 138) K2b1ck: 5′ GTCCCTGGATCCAGTGAG 3′ (Seq. ID No. 139) K3b1ck: 5′ CTCCCAGATACCACCGGA 3′ (Seq. ID No. 140) K3b1ck2: 5′ GCG CAG CTT CTC TTC CT 3′ (Seq. ID No.
- K3b1ck3 5′ CAC AGC TTC TTC TTC CTC 3′ (Seq. ID No. 142)
- K4b1ck 5′ ATCTCTGGTGCCTACGGG 3′ (Seq. ID No. 143)
- KSb1ck 5′ ATCTCTGATACCAGGGCA 3′ (Seq. ID No. 144)
- K6b1ck 5′ GTTCCAGCCTCCAGGGGT 3′ (Seq. ID No. 145)
- Example 3 The same protocol as Example 3 is employed using nesting oligonucleotides having the following sequences: Nesting oligonucleoties for Light Chain Kappa Vk1: HpK1L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GMC ATC CAG ATG ACC CAG TCT (Seq. ID No. 146) CCTAA 3′ wherein M is an equal mixture of A and C HpK1L2 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC AAC ATC CAG ATGACC CAG TCT (Seq. ID No.
- CCTAT 3′ Nesting oligonucleotides for Light Chain Kappa Vk2: HpK2L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAT ATT GIG ATG ACC CAG ACT (Seq. ID No. 151) CTTA 3′ HpK2L2 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAT GTT GTGATG ACT CAG TCT (Seq. ID No. 152) CC TAA 3′ HpK2L3 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAT ATT GIG AIG ACT GAG TCT (Seq. ID No.
- CCTAA 3′ Nesting oligonucleotides for Light Chain Kappa Vk3 HpK3L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAA ATT GTG TTG ACG GAG TCT (Seq. ID No. 154) CCTAA 3′ HpK3L2 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAA ATAGTG ATG ACG CAG TCT (Seq. ID No. 155) CCTAA 3′ HpK3L3 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAA ATT GTA ATG ACA GAG TCT (Seq. ID No.
- CCTAA 3′ Nesting oligonucleotides for Light Chain Kappa Vk4 HpK4L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAG ATC GTG AIG ACC GAG TCT (Seq. ID No. 157)
- CCTAT 3′ Nesting oligonucleotides for Light Chain Kappa Vk5 HpK5L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAA ACG ACA CTC ACG GAG TCT (Seq. ID No.
- CCTAA 3′ Nesting oligonucleotides for Light Chain Kappa Vk6: HpK6L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAA ATT GTG CTG ACT CAG TCT (Seq. ID No. 159) CCTAT 3′
- the same protocol as example 3 is employed using the following phosphoramidate boundary oligonucleotides designed to hybridize with the leader sequence of the lambda light chain genes.
- the phosphoramidate boundary oligonucleotides for lambda light chain genes have the sequences: PNL11d: 5′ CTG GGC CCA GGA CCC TGT GC 3′ (Seq. ID No. 160) PNL21d: 5′ CTG GGC CCA GGA CCC TGT 3′. (Seq. ID No. 161) PNL31d: 5′ GA GGC CAC AGA GCC TGT GCA GAG AGT GAG 3′ (Seq. ID No.
- PNL41d1 5′ CAG AGC ACA GAG ACC TGT GGA3′ (Seq. ID No. 163)
- PNL41d2 5′ CTG GGA GAG AGA CCC TGT CCA3′ (Seq. ID No. 164)
- PNL51d1 5′ CTG GGA GAG GGA ACC TGT GCA3′ (Seq. ID No. 165)
- PNL61d1 5′ ATT GGC CCA AGA ACC TGT GCA3′ (Seq. ID No. 166)
- PNL71d1 5′ CTG AGA ATT GGA CCC TGG GCA3′ (Seq. ID No.
- PNL81d1 5′ CTG AGA ATC CAC TCC TGA TCC3′
- PNL91d1 5′ CTG GGA GAG GGA CCC TGT 0A03′
- PNL101d1 5′ CTG GAC CAC TGA CAC TGC AGA3′ (Seq. ID No. 170)
- the same protocol as example 3 is employed using the following blocking check primers and primer CL2DX2, dNTPs, Advantage-2 DNA polymerase mix (Clontech), the reaction buffer, and the first strand cDNA synthesis product.
- the blocking check primers have the following sequences: L1b1ck: 5′ CAC TGY GCA GGG TCC TGG 3′ (Seq. ID No. 171) L2b1ck: 5′ CAG GGC ACA GGG TCC TGG 3′ (Seq. ID No. 172) L3b1ck1: 5′ TAC TGC ACA GGA TCC GTG 3′ (Seq. ID No.
- L3b1ck2 5′ CAC TTT ACA GGT TCT GTG 3′ (Seq. ID No. 174)
- L3b1ck3 5′ TTC TGC ACA GTC TCT GAG 3′ (Seq. ID No. 175)
- L3b1ck4 5′ CTC TGC ACA GGC TCT GAG 3′ (Seq. ID No. 176)
- L3b1ck5 5′ CTT TGC TCA GGT TCT GTG 3′ (Seq. ID No. 177)
- L3b1ck6 5′ CAC TGC ACA GGC TCT GTG 3′ (Seq. ID No.
- L3b1ck7 5′ CTC TAC ACA GGC TCT ATT 3′ (Seq ⁇ ID No. 179)
- L3b1ck7 5′ CTC TGC ACA GTC TCT GTG 3′ (Seq. ID No. 180)
- L4b1ck1 5′ TTC TCC ACA GGT CTC TGT 3′ (Seq. ID No. 181)
- L4b1ck2 5′ CAC TGG ACA GGG TCT CTC 3′ (Seq. ID No. 182)
- L5b1ck1 5′ CAC TGC ACA GGT TCC CTC 3′ (Seq. ID No.
- L6b1ck 5′ CAC TGC ACA GGT TCT TGG 3′ (Seq. ID No. 184)
- L7b1ck 5′ TGC TGC CCA GGG TCC AAT 3′ (Seq. ID No. 185)
- L8b1ck 5′ TAT GGA TCA GGA GTG GAT 3′ (Seq. ID No. 186)
- L9b1ck 5′ CTC CTC ACA GGG TCC CTC 3′ (Seq. ID No. 187)
- L10b1ck 5′ CAC TCT GCA GTG TCA GTG 3′ (Seq. ID No. 188)
- Example 3 The same protocol as Example 3 is employed using the nesting oligonucleotides having the following sequences: Nesting oligpnucleotides for Lambda Light Chain VL1: HpL1L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG TCT GTG CTG ACT GAG CCA (Seq. ID No. 190) CCAAA 3′ HpL1L2 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAG TCT GTG YTG ACG GAG CCG (Seq. ID No.
- GCAAA3′ Nesting oligonuele6tides for Lambda Light Chain VL6 HpL6L1 6a 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC AAT TTT ATG CTG ACT CAG CCC (Seq. ID No. 204)
- CAAAA3′ Nesting oligonucleotides for Lambda Light Chain VL7 and VL8 HpL7/8L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG ACT GTG GTG ACY CAG GAG (Seq. ID No.
- Example 4 The sample protocol as Example 4 was employed. Among the ninety-four clones screened eighty clones are positive.
- Example 4 The sample protocol as Example 4 was employed. Sequences of the variable regions of the heavy chain genes from fourteen positive clones are listed in FIG. 6. The sequence diversity of these clones and others produced shows this method can efficiently amplify the repertoire of human heavy chain genes from immunized donors.
Abstract
Templates that are engineered to contain a predetermined sequence and a hairpin structure are provided by a nested oligonucleotide extension reaction. The engineered template allows Single Primer Amplification (SPA) to amplify a target sequence within the engineered template. In particularly useful embodiments, the target sequences from the engineered templates are cloned into expression vehicles to provide a library of polypeptides or proteins, such as, for example, an antibody library.
Description
- This application claims priority to U.S. Provisional Application No. 60/254,669 filed Dec. 11, 2000 and to U.S. Provisional Application No. 60/323,400 filed Sep. 19, 2001. The disclosures of both these Provisional applications are incorporated herein in their entirety by this reference.
- This disclosure relates to engineered templates useful for amplification of a target nucleic acid sequence. More specifically, templates which are engineered to contain a predetermined sequence and a hairpin structure are provided by a nested oligonucleotide extension reaction. The engineered templates allow Single Primer Amplification (SPA) to amplify a target sequence within the engineered template. In particularly useful embodiments, the target sequences from the engineered templates are cloned into expression vehicles to provide a library of polypeptides or proteins, such as, for example, an antibody library.
- Methods for nucleic acid amplification and detection of amplification products assist in the detection, identification, quantification, isolation and sequence analysis of nucleic acid sequences. Nucleic acid amplification is an important step in the construction of libraries from related genes such as, for example, antibodies. These libraries can be screened for antibodies having specific, desirable activities. Nucleic acid analysis is important for detection and identification of pathogens, detection of gene alteration leading to defined phenotypes, diagnosis of genetic diseases or the susceptibility to a disease, assessment of gene expression in development, disease and in response to defined stimuli, as well as the various genome projects. Other applications of nucleic acid amplification method include the detection of rare cells, detection of pathogens, and the detection of altered gene expression in malignancy, and the like. Nucleic acid amplification is also useful for qualitative analysis (such as, for example, the detection of the presence of defined nucleic acid sequences) and quantification of defined gene sequences (useful, for example, in assessment of the amount of pathogenic sequences as well as the determination of gene multiplication or deletion, and cell transformation from normal to malignant cell type, etc.). The detection of sequence alterations in a nucleic acid sequence is important for the detection of mutant genotypes, as relevant for genetic analysis, the detection of mutations leading to drug resistance, pharmacogenomics, etc.
- There are many variations of nucleic acid amplification, for example, exponential amplification, linked linear amplification, ligation-based amplification, and transcription-based amplification. One example of exponential nucleic acid amplification method is polymerase chain reaction (PCR) which has been disclosed in numerous publications. See, for example, Mullis et al. Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Mullis K. EP 201,184; Mullis et al. U.S. Pat. No. 4,582,788; Erlich et al. EP 50,424, EP 84,796, EP 258,017, EP 237,362; and Saiki R. et al. U.S. Pat. No. 4,683,194. In fact, the polymerase chain reaction (PCR) is the most commonly used target amplification method. PCR is based on multiple cycles of denaturation, hybridization of two different oligonucleotide primers, each to opposite strand of the target strands, and primer extension by a nucleotide polymerase to produce multiple double stranded copies of the target sequence.
- Amplification methods that employ a single primer, have also been disclosed. See, for example, U.S. Pat. Nos. 5,508,178; 5,595,891; 5,683,879; 5,130,238; and 5,679,512. The primer can be a DNA/RNA chimeric primer, as disclosed in U.S. Pat. No. 5,744,308.
- Some amplification methods use template switching oligonucleotides (TSOs) and blocking oligonucleotides. For example, a template switch amplification in which chimeric DNA primer are utilized is disclosed in U.S. Pat. Nos. 5,679,512; 5,962,272; 6,251,639; and by Patel et al. Proc. Natl. Acad. Sci. U.S.A. 93:2969-2974 (1996).
- However the previously described target amplification methods have several drawbacks. For example, the transcription base amplification methods, such as Nucleic Acid Sequence Based Amplification (NASBA) and transcription mediated amplification (TMA), are limited by the need for incorporation of the polymerase promoter sequence into the amplification product by a primer, a process prone to result in non-specific amplification. Another example of a drawback of the current amplification methods is the requirement of two binding events which may have optimal binding at different temperatures. This combination of factors results in increased likelihood of mis-priming and resultant amplification of sequences other than the target sequence. Therefore, there is a need for improved nucleic acid amplification methods that overcome these drawbacks. The invention provided herein fulfills this need and provides additional benefits.
- A method of amplifying nucleic acid has been discovered which includes the steps of a) annealing a primer to a template nucleic acid sequence, the primer having a first portion which anneals to the template and a second portion of predetermined sequence; b) synthesizing a polynucleotide that anneals to and is complementary to the portion of the template between the location at which the first portion of the primer anneals to the template and the end of the template, the polynucleotide having a first end and a second end, wherein the first end incorporates the primer; c) separating the polynucleotide synthesized in step (b) from the template; d) annealing a nested oligonucleotide to the second end of the polynucleotide synthesized in step (b), the nested oligonucleotide having a first portion that anneals to the second end of the polynucleotide, and a second portion having a hairpin structure; e) extending the polynucleotide synthesized in step (b) to provide a portion that is complimentary to the hairpin structure and a terminal portion that is complementary to the predetermined sequence; and f) amplifying the extended polynucleotide using a single primer having the predetermined sequence.
- In an alternative embodiment, the method of amplifying nucleic acid includes the steps of a) annealing a primer and a boundary oligonucleotide to a template nucleic acid sequence, the primer having a first portion which anneals to the template and a second portion of predetermined sequence; b) synthesizing a polynucleotide that anneals to and is complementary to the portion of the template between the location at which the first portion of the primer anneals to the template and the portion of the template to which the boundary oligonucleotide anneals, the polynucleotide having a first end and a second end, wherein the first end incorporates the primer; c) separating the polynucleotide synthesized in step (b) from the template; d) annealing a nested oligonucleotide to the second end of the polynucleotide synthesized in step (b), the nested oligonucleotide having a first portion that anneals to the second end of the polynucleotide and a second portion having a hairpin structure; e) extending the polynucleotide synthesized in step (b) to provide a portion that is complementary to the hairpin structure and a terminal portion that is complementary to the predetermined sequence; and f) amplifying the extended polynucleotide using a single primer having the predetermined sequence.
- In yet another embodiment, the method of amplifying nucleic acid includes the steps of a) annealing an oligo dT primer and a boundary oligonucleotide to an mRNA template; b) synthesizing a polynucleotide that anneals to and is complementary to the portion of the template between the location at which the first portion of the primer anneals to the template and the portion of the template to which the boundary oligonucleotide anneals, the polynucleotide having a first end and a second end, wherein the first end incorporates the primer; c) separating the polynucleotide synthesized in step (b) from the template; d) annealing a nested oligonucleotide to the second end of the polynucleotide synthesized in step (b), the nested oligonucleotide having a first portion that anneals to the second end of the polynucleotide, and a second portion having a hairpin structure; e) extending the polynucleotide synthesized in step (b) to provide an extended polynucleotide that includes a portion that is complementary to the hairpin structure and a poly A terminal portion; and f) amplifyng the extended polynucleotide using a single primer.
- In another aspect an engineered nucleic acid strand is disclosed which has a predetermined sequence at a first end thereof, a sequence complementary to the predetermined sequence at the other end thereof, and a hairpin structure therebetween.
- In yet another aspect, a method of amplifying a nucleic acid strand has been discovered which includes the steps of providing an engineered nucleic acid strand having a predetermined sequence at a first end thereof, a sequence complementary to the predetermined sequence at the other end thereof and a hairpin structure therebetween, and contacting the engineered nucleic acid strand with a primer containing at least a portion of the predetermined sequence in the presence of a polymerase and nucleotides under conditions suitable for polymerization of the nucleotides.
- Once the engineered nucleic acid is amplified a desired number of times, restriction sites can be used to digest the strand so that the target nucleic acid sequence can be ligated into a suitable expression vector. The vector may then be used to transform an appropriate host organism using standard methods to produce the polypeptide or protein encoded by the target sequence. In particularly useful embodiments, the techniques described herein are used to amplify a family of related sequences to build a complex library, such as, for example, an antibody library.
- FIG. 1 is a schematic illustration of a primer and boundary oligo annealed to a template;
- FIG. 2A is a schematic illustration of a restriction oligo annealed to a nucleic acid strand;
- FIG. 2B is a schematic illustration of a primer annealed to a template that has a shortened 5′ end;
- FIG. 3 is a schematic illustration of a nested oligo having a hairpin structure annealed to a newly synthesized nucleic acid strand;
- FIG. 4A is a schematic illustration of an engineered template in accordance with this disclosure; and
- FIG. 4B is a schematic illustration of an engineered template in accordance with an alternative embodiment.
- FIGS.5A-5C is a chart showing the sequences of clones produced in Example 4.
- FIGS.6A-6C is a chart showing the sequences of clones produced in Example 7.
- The present disclosure provides a method of amplifying a target nucleic acid sequence. In particularly useful embodiments, the target nucleic acid sequence is a gene encoding a polypeptide or protein. The disclosure also describes how the products of the amplification may be cloned and expressed in suitable expression systems. In particularly useful embodiments, the techniques described herein are used to amplify a family of related sequences to build a complex library, such as, for example, an antibody library.
- The target nucleic acid sequence is exponentially amplified through a process that involves only a single primer. The ability to employ a single primer (i.e., without the need for both forward and reverse primers each having different sequences) is achieved by engineering a strand of nucleic acid that contains the target sequence to be amplified. The engineered strand of nucleic acid (sometimes referred to herein as the “engineered template”) is prepared from two templates; namely, 1) a starting material that is a natural or synthetic nucleic acid (e.g., RNA, DNA or cDNA) containing the sequence to be amplified and 2) a nested oligonucleotide that provides a hairpin structure. The starting material can be used directly as the original template, or, alternatively a strand complementary to the starting material can be prepared and used as the original template. The nested oligonucleotide is used as a template to extend the nucleotide sequence of the original template during creation of the engineered strand of nucleic acid. The engineered strand of nucleic acid is created from the original template by a series of manipulations that result in the presence of a predetermined sequence at one end thereof and a hairpin structure. It is these two features that allow amplification using only a single primer.
- Any nucleic acid, in purified or nonpurified form, can be utilized as the starting material for the processes described herein provided it contains or is suspected of containing the target nucleic acid sequence to be amplified. Thus, the starting material employed in the process may be, for example, DNA or RNA, including messenger RNA, which DNA or RNA may be single stranded or double stranded. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized. A mixture of any of these nucleic acids may also be employed, or the nucleic acids produced from a previous amplification reaction herein using the same or different primers may be utilized. The target nucleic acid sequence to be amplified may be a fraction of a larger molecule or can be present initially as a discrete molecule. The starting nucleic acid may contain more than one desired target nucleic acid sequence which may be the same or different. Therefore, the present process may be useful not only for producing large amounts of one target nucleic acid sequence, but also for amplifying simultaneously more than one different target nucleic acid sequence located on the same or different nucleic acid molecules.
- The nucleic acids may be obtained from any source, for example: genomic or cDNA libraries, plasmids, cloned DNA or RNA, or from natural DNA or RNA from any source, including bacteria, yeast, viruses, and higher organisms such as plants or animals. The nucleic acid can be naturally occurring or synthetic, either totally or in part. Techniques for obtaining and producing the nucleic acids used in the present processes are well known to those skilled in the art. If the nucleic acid contains two strands, it is necessary to separate the strands of the nucleic acid before it can be used as the original template, either as a separate step or simultaneously with the synthesis of the primer extension products. Additionally, if the starting material is first strand DNA, second strand DNA may advantageously be created by processes within the purview of those skilled in the art and used as the original template from which the engineered template is created.
- First strand cDNA and mRNA are particularly useful as the original template for the present methods. Suitable methods for generating DNA templates are known to and readily selected by those skilled in the art. In one embodiment, 1st strand cDNA is synthesized in a reaction where reverse transcriptase catalyzes the synthesis of DNA complementary to any RNA starting material in the presence of an oligodeoxynucleotide primer and the four deoxynucleoside triphosphates, dATP, dGTP, dCTP, and TTP. The reaction is initiated by the non-covalent bonding of the oligo-deoxynucleotide primer to the 3′ end of mRNA followed by stepwise addition of the appropriate deoxynucleotides as determined by base pairing relationships with the mRNA nucleotide sequence, to the 3′ end of the growing chain. As those skilled in the art will appreciate, all mRNA in a sample can be used to generate first strand cDNA through the annealing of oligo dT to the poly A tail of the mRNA.
- Once the original template is obtained, a
primer 20 and aboundary oligonucleotide 30 are annealed to theoriginal template 10. (See FIG. 1.) A strand of nucleic acid complementary to the portion of the original template beginning at the 3′ end of the primer up to about the 5′ end of the boundary oligonucleotide is polymerized. - The
primer 20 that is annealed to the original template includes a portion 25 that anneals to the original template and optionally aportion 22 of predetermined sequence that preferably does not anneal to the template, and optionally arestriction site 23 betweenportions 22 and 25. Thus, for example, where the original template is mRNA, a portion having a predetermined sequence that does not anneal to the template is not needed, but rather the primer can be any gene-specific internal sequence of the mRNA or oligo dT which will anneal to the unique poly A tail of the mRNA. - The primer anneals to the original template adjacent to the
target sequence 12 to be amplified. It is contemplated that the primer can anneal to the original template upstream of the target sequence (or downstream in the case, e.g., of an mRNA original template) to be amplified, or that the primer may overlap the beginning of thetarget sequence 12 to be amplified as shown in FIG. 1. The predetermined sequence ofportion 22 of the primer is selected so as to provide a sequence to which the single primer used during the amplification process can hybridize as described in detail below. Preferably, the predetermined sequence is not native in the original template and does not anneal to the original template, as shown in FIG. 1. Optionally, the predetermined sequence may include a restriction site useful for insertion of a portion of the engineered template into an expression vector as described more fully hereinbelow. - The
boundary oligonucleotide 30 that is annealed to the original template serves to terminate polymerization of the nucleic acid. Any oligonucleotide capable of terminating nucleic acid polymerization may be utilized as theboundary oligonucleotide 30. In a preferred embodiment the boundary oligonucleotide includes afirst portion 35 that anneals to theoriginal template 10 and asecond portion 32 that is not susceptible to an extension reaction. Techniques to prevent the boundary oligo from acting as a site for extension are within the purview of one skilled in the art. By way of example,portion 32 of theboundary oligo 30 may be designed so that it does not anneal to theoriginal template 10 as shown in FIG. 1. In such embodiments, theboundary oligonucleotide 30 prevents further polymerization but does not serve as a primer for nucleic acid synthesis because the 3′ end thereof does not hybridize with theoriginal template 10. Alternatively, the 3′ end of theboundary oligo 30 might be designed to include locked nucleic acid to achieve the same effect. Locked nucleic acid is disclosed for example in WO 99/14226, the contents of which are incorporated herein by reference. Those skilled in the art will envision other ways of ensuring that no extension of the 3′ end of the boundary oligo occurs. - Primers and oligonucleotides described herein may be synthesized using established methods for oligonucleotide synthesis which are well known in the art. Oligonucleotides, including primers of the present invention include linear oligomers of natural or modified monomers or linkages, such as deoxyribonucleotides, ribonucleotides, and the like, which are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to monomer interactions such as Watson-Crick base pairing. Usually monomers are linked by phosphodiester bonds or their analogs to form oligonucleotides ranging in size from a few monomeric units e.g., 3-4, to several tens of monomeric units. A primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers known in the art may be useful for the methods of the present disclosure. A primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers may be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
- Polymerization of nucleic acid can be achieved using methods known to those skilled in the art. Polymerization is generally achieved enzymatically, using a DNA polymerase or reverse transcriptase which sequentially adds free nucleotides according to the instructions of the template. The selection of a suitable enzyme to achieve polymerization for a given template and primer is within the purview of those skilled in the art. In certain embodiments, the criteria for selection of polymerases includes lack exonuclease activity or DNA polymerases which do not possess a strong exonuclease activity. DNA polymerases with low exonuclease activity for use in the present process may be isolated from natural sources or produced through recombinant DNA techniques. Illustrative examples of polymerases that may be used, are, without limitation, T7 Sequenase v. 2.0, the Klenow Fragment of DNA polymerase I lacking exonuclease activity, the Klenow Fragment of Taq Polymerase, exo.-Pfu DNA polymerase, Vent. (exo.-) DNA polymerase, and Deep Vent. (exo-) DNA polymerase.
- In a particularly useful embodiment, the use of a boundary oligonucleotide is avoided by removing unneeded portions of the starting material by digestion. In this embodiment, which is shown schematically in FIG. 2A, a
restriction oligonucleotide 70 is annealed to the starting material 100 at a preselected location. The restriction oligonucleotide provides a double stranded portion on the starting material containing arestriction site 72. Suitable restriction sites, include, but are not limited to Xho I, Spe I, Nhe1, Hind III, Nco I, Xma I, Bg1 II, Bst I, and Pvu I. Upon exposure to a suitable restriction enzyme, the starting material is digested and thereby shortened to remove unnecessary sequence while preserving the desired target sequence 12 (or portion thereof) to be amplified on what will be used as theoriginal template 110. Once theoriginal template 110 is obtained, aprimer 20 is annealed to the original template 110 (see FIG. 2B) adjacent to or overlapping with thetarget sequence 12 as described above in connection with previous embodiments. A strand ofnucleic acid 40 complementary to the portion of the original template between the 3′ end of theprimer 20 and the 5′ end of theoriginal template 110 is polymerized. As those skilled in the art will appreciate, in this embodiment where a restriction oligonucleotide is employed to generate the original template, there is no need to use a boundary oligonucleotide, because primer extension can be allowed to proceed all the way to the 5′ end of the shortenedoriginal template 110. - Once polymerization is complete (i.e., growing
strand 40 reaches theboundary oligonucleotide 30 or the 5′ end of the shortened original template 110), the newly synthesized complementary strand is separated from the original template by any suitable denaturing method including physical, chemical or enzymatic means. Strand separation may also be induced by an enzyme from the class of enzymes known as helicases or the enzyme RecA, which has helicase activity and in the presence of riboATP is known to denature DNA. The reaction conditions suitable for separating the strands of nucleic acids with helicases are described by Cold Spring Harbor Symposia on Quantitative Biology, Vol. XLIII “DNA: Replication and Recombination” (New York: Cold Spring Harbor Laboratory, 1978), B. Kuhn et al., “DNA Helicases”, pp. 63-67, and techniques for using RecA are reviewed in C. Radding, Ann. Rev. Genetics, 16:405-37 (1982). - The newly synthesized complementary strand thus includes sequences provided by the primer20 (e. g., the
predetermined sequence 22, theoptional restriction site 23 and the annealing portion 25 of the primer) as well as the newly synthesizedportion 45 that is complementary to the portion of theoriginal template 10 between the location at which theprimer 20 was annealed to theoriginal template 10 and either the portion of theoriginal template 10 to which theboundary oligonucleotide 30 was annealed or through to the shortened 5′ end of the original template. See FIG. 3. - Optionally, multiple rounds of polymerization using the original template and a primer are performed to produce multiple copies of the newly synthesized complementary strand for use in subsequent steps. It is contemplated that 2 to 10 rounds or more (preferably, 15-25 rounds) of linear amplification can be performed when a DNA template is used. Making multiple copies of the newly synthesized complementary strand at this point in the process (instead of waiting until the entire engineered template is produced before amplifying) helps ensure that accurate copies of the target sequence are incorporated into the engineered templates ultimately produced. It is believed that multiple rounds of polymerization based on the original template provides a greater likelihood that a better representation of all members of the library will be achieved, therefore providing greater diversity compared to a single round of polymerization.
- The next step in preparing the engineered template involves annealing a nested
oligonucleotide 50 to the 3′ end of the newly synthesized complementary strand, for example as shown in FIG. 3. As seen in FIG. 3, the nestedoligonucleotide 50 provides a template for further polymerization necessary to complete the engineered template.Nested oligonucleotide 50 includes aportion 52 that does not hybridize and/or includes modified bases to the newly synthesized complementary strand, thereby preventing the nested oligonucleotide from serving as a primer.Nested oligonucleotide 50 also includes aportion 55 that hybridizes to the 3′ end of the newly synthesized complementary strand.Nested oligonucleotide 50 may optionally also define arestriction site 56. The final portion 58 of nestedoligonucleotide 50 contains a hairpin structure. From the point at whichportion 55 extends beyond the 3′ end of the beginning the newly synthesized complementary strand, the nested oligonucleotide serves as a template for further polymerization to form the engineered template. It should be understood that the nested oligo may contain part of the target sequence (if part thereof was truncated in forming the original template) or may include genes that encode a polypeptide or protein (or portion thereof) such as, for example, one or more CDR's or Framework regions or constant regions of an antibody. It is also contemplated that a collection of nested oligonucleotides having different sequences can be employed, thereby providing a variety of templates which results in a library of diverse products. Thus, polymerization will extend the newly synthesized complementary strand by adding additionalnucleic acid 60 that is complementary to the nested oligonucleotide as shown in FIG. 3. Techniques for achieving polymerization are within the purview of one skilled in the art. As previously noted, in selecting a suitable polymerase, an enzyme lacking exonuclease activity may be employed to prevent the 3′ end of the nested oligo from acting as a primer. Because ofhairpin structure 50 of the nested oligonucleotide, eventually the newly synthesized complementary strand will turn back ontoportion 45 of the same strand which will then serve as the template for further polymerization. Polymerization will continue until the end of the primer is reached, at which point the newly synthesized strand will terminate with a portion whose sequence is complementary to the primer. - Once polymerization is complete, the engineered
template 120 is separated from the nestedoligonucleotide 50 by techniques well known to those skilled in the art such as, for example, heat denaturation. The resulting engineeredtemplate 120 contains a portion derived from theoriginal primer 20,portion 45 that is complementary to a portion of the original template, andportion 65 that is complementary to a portion of the nested oligonucleotide and includes ahairpin structure 68, and aportion 69 that is complementary toportion 45. (See FIGS. 4A and B.) The 3′ end of engineeredtemplate 120 includes a portion containing a sequence that is complementary toprimer 20. Thus, for example, as shown in FIG. 4A, the 3′ end of engineeredtemplate 120 includesportion 22′ containing a sequence that is complementary to the predetermined sequence ofportion 22 ofprimer 20. This allows for amplification of the desired sequence contained within engineeredtemplate 120 using a single primer having the same sequence as the predetermined sequence of primer portion 22 (or a primer that is complementary thereto) using techniques known to those of ordinary skill in the art. - As another example (shown in FIG. 4B), where mRNA is used as the template and oligo dT is used as the primer, the 3′ end of engineered
template 120 includes poly A portion that is complementary to the oligo dT primer. In this case, any sequence alongportion 45 can be selected for use as the primer to be annealed toportion 69 once the engineered template is denatured for single primer amplification. Optionally, the primer may include a non-annealing portion, such as, for example, a portion defining a restriction site. - During single primer amplification, the presence of a polymerase having exonuclease activity is preferred because such enzymes are known to provide a “proofreading” function and have relatively higher processivity compared to polymerases lacking exonuclease activity.
- Due to
hairpin structure 68 there is internal self annealing between the 5′ end predetermined sequence and the 3′ end sequence which is complementary to the predetermined sequence on the engineered template. Upon denaturation and addition of a primer having the predetermined sequence, the primer will hybridize to the template and amplification can proceed. - After amplification is performed, the products may be detected using any of the techniques known to those skilled in the art. Examples of methods used to detect nucleic acids include, without limitation, hybridization with allele specific oligonucleotides, restriction endonuclease cleavage, single-stranded conformational polymorphism (SSCP), analysis gel electrophoresis, ethidium bromide staining, fluorescence resonance energy transfer, hairpin FRET essay, and TaqMan assay.
- Once the engineered nucleic acid is amplified a desired number of times,
restriction sites - In particularly useful embodiments, the methods described herein are used to amplify target sequences encoding antibodies or portions thereof, such as, for example the variable regions (either light or heavy chain) using cDNA of an antibody. In this manner, a library of antibodies can be amplified and screened. Thus, for example, starting with a sample of antibody mRNA that is naturally diverse, first strand cDNA can be produced and digested to provide an original template. A primer can be designed to anneal upstream to a selected complementary determining region (CDR) so that the newly synthesized nucleic acid strand includes the CDR. By way of example, if the target sequence is heavy chain CDR3, the primer may be designed to anneal to the heavy chain framework one (FR1) region. Those skilled in the art will readily envision how to design appropriate primers to anneal to other upstream sites or to reproduce other selected targets within the antibody cDNA based on this disclosure.
- The following Examples are provided to illustrate, but not limit, the present invention(s):
- First Strand cDNA Synthesis
- First strand cDNA to be used as the original template was generated from 2 μg of human peripheral blood lymphocyte (PBL) mRNA with an oligo-dT primer using the SuperScript II First Strand Synthesis Kit (Invitrogen) according to the manufacturer's instructions. The 1st strand cDNA product was purified over a QIAquick spin column (QIAGEN PCR Purification Kit) and eluted in 400 μL of nuclease-free water.
- Second Strand Linear Amplification (SSLA) in the Presence of Blocking Oligonucleotide
- The second strand cDNA reaction contained 5 μL of 1st strand cDNA original template, 0.5 μM primer JMX26VK1a, 0.5 μM blocking oligo CKLNA1, 0.2 mM dNTPs, 5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems), 1× GeneAmp Gold Buffer (15 Mm Tris-HCl, pH 8.0, 50 mM KCl), and 1.5 mM MgCl2. The final volume of the reaction was 98 μL. The sequence of primer JMX26VK1a, which hybridizes to the
framework 1 region of VK1a genes, was 5′ GTC ACT CAC GAA CTC ACG ACT CAC GGA GAG CTC RAC ATC CAGATG ACC CAG 3′ (Seq. ID No. 1) where R is an equal mixture of A and G. The sequence of the blocking oligo CKLNA1, which hybridizes to the 5′ end of the VK constant region, was 5′ GAA CTG TGG CTGCAC CAT CTG 3′ (Seq. ID No. 2), where the underlined bases are locked nucleic acid (LNA) nucleotide analogues. After an initial heat denaturation step of 94° C. for 3 minutes, linear amplification of 2nd strand cDNA was carried out for 20 cycles of 94° C. for 15 seconds, 56° C. for 15 seconds, and 68° C. for 1 minute. - Nested Oligo Extension Reaction
- After the last cycle of linear amplification, 2 μL of a nested/hairpin oligo designated “JK14TSHP” was added to give a final concentration of 20 μM. The sequence of JK14TSHP was 5′ CCT TAG AGT CAC GCT AGC GAT TGA TTG ATT GAT TGATTG TTT GTG ACT CTA AGG TTG GCG CGC CTT CGT TTG ATY TCC ACC TTG GTC C(ps)T(ps)G(ps)
P 3′ (Seq. ID No. 3) where Y is an equal mixture of C and T and (ps) are phosphorothioate backbone linkages and P is a 3′ propyl group. For nested oligo extension reaction, two cycles of 94° C. for 1 minute, 56° C. for 15 seconds, and 72° C. for 1 minute were performed, followed by a 10 minute incubation at 72° C. to allow complete extension of the hairpin. The reaction products were purified over a QIAquick spin column (QIAgen PCR Purification Kit) and eluted in 50 μL of nuclease-free water. - Analysis of Engineered Template
- The efficiency of the nested oligo extension reaction was determined by amplifying the products with either a primer set specific for the engineered product or a primer set that detects all VK1a/JK14 second strand cDNA products (including the engineered product). For specific detection of engineered product, a 10 μL aliquot was amplified for 20 or 25 cycles with primers designated “JMX26” and “TSDP”. Primer JMX26 hybridizes to the 5′ end of JMX26VK1a, the
framework 1 primer used in the second strand cDNA reaction. Primer TSDP hybridizes to the hairpin-loop sequence added to the 3′ ends of the second strand cDNAs in the nested oligo extension reaction. The sequence of primer JMX26 was 5′ GTC ACT CAC GAA CTC ACGACT CAC GG 3′ (Seq. ID No. 4). The sequence of primer TSDP was 5′ CAC GCT AGC GAT TGATTG ATT G 3′ (Seq. ID No. 5). For detection of all VK1a/JK14 second strand cDNA products a 10 μL aliquot was amplified for 20 or 25 cycles with primers JMX26 and JK14. The sequence of primer JK14, which hybridizes to the framework 4 region of JK1 and JK4 genes, was 5′ GAG GAG GAG GAG GAG GAG GGC GCG CCT GAT YTC CACCTT GGT CCC 3′ (Seq. ID No. 6). Both reactions contained 1× GeneAmp Gold Buffer, 1.5 mM MgCl2, 7.5% glycerol, 0.2 mM dNTPs, and 0.5 μM of each primer in a final volume of 50 μL. - The results with primers JMX26 and TSDP demonstrated the successful production of nested oligo and extended VK stem-loop DNA when using SSLA DNA that was blocked specifically with a boundary oligo. Suitable controls showed that when using the nested oligo in the presence of SSLA DNA that was not blocked, only a minimal amount of amplified product was produced. Additional controls without the nested oligo were negative. However, VK1a/JK14 second strand cDNA products were detected equally among all tested samples.
- Single Primer Amplification of the Stem-loop cDNA Template
- Conditions that were previously shown to amplify a 352 bp stem-15 bp loop DNA product were as follows: 10 pg of the stem-loop DNA, 2 μM primer, 50 mM Tris-HCl, pH 9.0, 1.5 mM MgCl2, 15 mM (NH4)2SO4, 0.1% Triton X-100, 1.7 M betaine, 0.2 mM dNTPs, and 2.5 units of Z-Taq DNA Polymerase (Takara Shuzo) in a final volume of 50 μL. The thermal cycling conditions were an initial denaturation step of 96° C. for 2.5 minutes, 35 cycles of 96° C. for 30 seconds, 64° C. for 30 seconds, 74° C. for 1.5 minutes, and a final extension step of 74° C. for 10 minutes. Oligonucleotides containing the modified bases 5-methyl-2′-deoxycytidine and/or 2-amino-2′-deoxyadenosine have been shown to prime much more efficiently than unmodified oligonucleotides at primer binding sites located within hairpin structures (Lebedev et al. 1996. Genetic Analysis: Biomolecular Engineering 13, 15-21). These modifications work by increasing the melting temperature of the primer, allowing the annealing step of the amplification to be performed at a higher temperature. JMX26 primers containing ten 5-methyl-2′-deoxycytidines or seven 2-amino -2′-deoxyadenosines have been synthesized.
- Cloning VK Products
- Amplified fragments are cloned by Sac I/Asc I into an appropriate expression vector that contains, in frame, the remaining portion of the kappa constant region. Suitable vectors include pRL5 and pRL4 vectors (described in
U.S. Provisional Application 60/254,411, the disclosure of which is incorporated hererin by reference), fdtetDOG, PHEN1, and pCANTAB5E. Individual kappa clones can be sequenced. - Expanding the Repertoire of VKappa Amplified Products
- Further coverage of the VK repertoire is achieved by using the above protocols with a panel of primers for the generation of the second strand DNA. The primers contain JMX26 sequence, a Sac I restriction site, and a region that anneals to 1st strand cDNA in the
framework 1 region of human antibody kappa light chain genes. The antibody annealing sequences were derived from the VBase database primers (www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html) which were designed based on the known sequences of human antibodies and are reported to cover the entire human antibody repertoire of kappa light chain genes. Below is a list of suitable primers:JMX26Vk1a GTCACTCACGAACTCACGACTCACGGAGAGCTCRACATCCAGATGACCCAG (Seq. ID No.7) JMX26Vk1b GTCACTCACGAACTCACGACTCACGGAGAGCTCGMCATCCAGTTGACCCAG (Seq. ID No.8) JMX26Vk1c GTCACTCACGAACTCACGACTCACGGAGAGCTCGCCATCCRGATGACCCAG (Seq. ID No.9) JMX2GVk1d GTCACTCACGAACTCACGACTCACGGAGAGCTCGTCATCTGGATGACCCAG (Seq. ID No.10) JMX26Vk2a GTCACTCACGAACTCACGACTCACGGAGAGCTCGATATTGTGATGACCCAG (Seq. ID No.11) JMX26Vk2b GTCACTCACGAACTCACGACTCACGGAGAGCTCGATRTTGTGATGACTCAG (Seq. ID No.12) JMX26Vk3a GTCACTCACGAACTCACGACTCACGGAGAGCTCGAAATTGTGTTGACRCAG (Seq. ID No.13) JMX26Vk3b GTCACTCACGAACTCACGACTCACGGAGACCTCGAAATAGTGATGACGCAG (Seq. ID No.14) JMX2GVk3c GTCACTCACGAACTCACGACTCACGGAGAGCTCGAAATTGTAATGACACAG (Seq. ID No.15) JMX26Vk4a GTCACTCACGAACTCACGACTCACGGAGAGCTCGACATCGTGATGACCCAG (Seq. ID No.16) JMX26Vk5a GTCACTCACGAACTCACGACTCACGGAGAGCTCGAAACGACACTCACGCAG (Seq. ID No.17) JMX2GVk6a GTCACTCACGAACTCACGACTCACGGAGAGCTCGAAATTGTGCTGACTCAG (Seq. ID No.18) JMX2Gvk6b GTCACTCACGAACTCACGACTCACGGAGAGCTCGATGTTGTGATGACACAG (Seq. ID No.19) - In the foregoing sequences, R is an equal mixture of A and G, M is an equal mixture of A and C, Y is an equal mixture of C and T, W is an equal mixture of A and T, and S is an equal mixture of C and G.
- Similar protocols are applied to the amplification of both heavy chain and lambda light chain genes. JMX26, or another primer without antibody specific sequences, is used for each of those applications. If JMX26 is used, the second strand DNA is generated with the primers listed below which contain JMX26 sequence, a restriction site (Sac I for lambda, Xho I for heavy chains), and a region that anneals to 1st strand cDNA in the
framework 1 region of human antibody lambda light chain or heavy chain genes. The antibody annealing sequences were derived from the VBase database primers (www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html) which were designed based on the known sequences of human antibodies and are reported to cover the entire human antibody repertoire of lambda light chain and heavy chain genes. - Lambda
Light Chain Framework 1 Specific Primers:JMX2GVL1a GTCACTCACGAACTGACGACTCACGGAGAGCTCCAGTCTGTGCTGACTCAG (Seq. ID No. 20) JMX2GVL1b GTCACTCACGAACTCACGACTCACGGAGAGCTCCAGTCTGTGYTGACGCAG (Seq. ID No. 21) JMX264VL1C CTCACTCACGAACTCACGACTCACGGAGAGCTCCAGTCTGTCGTGACGCAG (Seq. ID No. 22) JMX26VL2 GTCACTCACGAACTCACGACTCACGGAGAGCTCCAGTCTGCCCTGACTCAG (Seq. ID No. 23) JMX26VL3a GTCACTCACGAACTCACCACTCACGGAGAGCTCTCCTATGWGCTGACTCAG (Seq. ID No. 24) JMX26VL3b GTCACTCACGAACTCACGACTCACGGAGAGCTCTCCTATGAGCTGACACAG (Seq. ID No. 25) JMX26VL3c GTCACTCACGAACTCACGACTCACGGAGAGCTCTCTTCTGAGCTGACTCAG (Seq. ID No. 26) JMX26VL3d GTCACTCACGAACTCACGACTCACGGAGAGCTCTCCTATGAGCTGATGCAG (Seq. ID No. 27) JMX26VL4 GTCACTCACGAACTCACCACTCACGGAGAGCTCCAGCYTGTGCTGACTCAA (Seq. ID No. 28) JMX26VL5 GTCACTCACGAACTCACCACTCACGGAGAGCTCCACSCTGTGCTGACTCAG (Seq. ID No. 29) JMX26VL6 GTCACTCACGAACTCACGACTCACGGAGAGCTCAATTTTATGCTGACTCAG (Seq. ID No. 30) JMX26VL7 GTCACTCACGAACTCACGACTCACGGAGAGCTCCAGRCTGTGGTGACTCAG (Seq. ID No. 31) JMX26VL8 GTCACTCACGAACTCACGACTCACGGAGAGCTCCAGACTGTGGTGACCCAG (Seq. ID No. 32) JMX26VL4/9 GTCACTCACGAACTCACGACTCACGGAGAGCTCCWGCCTGTGCTGACTCAG (Seq. ID No. 33) JMX26VL10 GTCACTCACGAACTCACGACTCACGGAGAGCTCCAGGCAGGGCTGACTCAG (Seq. ID No. 34) - In the foregoing sequences (and throughout this disclosure), R is an equal mixture of A and G, M is an equal mixture of A and C, Y is an equal mixture of C and T, W is an equal mixture of A and T, and S is an equal mixture of C and G.
-
Heavy Chain Framework 1 Specific Primers:JMX24VH1a GTCACTCACGAACTCACGACTCACGGActcgagCAGGTKCAGCTGGTGCAG (Seq. ID No. 35) JMX24VH1b GTCACTCACGAACTCACGACTCACGGActcgagCAGGTCCAGCTTGTGCAG (Seq. ID No. 36) JMX26VH1c GTCACTCACGAACTCACGACTCACGGActcgagSAGGTCCAGCTGGTACAG (Seq. ID No. 37) JMX26VH1d GTCACTCACGAACTCACGACTCACGGActcgagCARATGCAGCTGGTGCAG (Seq. ID No. 38) JMX26VH2a GTCACTCACGAACTCACGACTCACGGACtcgagCAGATCACCTTGAAGGAG (Seq. ID No. 39) JMX26VH2b GTCACTCACGAACTCACGACTCACGGActcgagCAGGTCACCTTGARGGAG (Seq. ID No. 40) JMX26VH3a GTCACTCACGAACTCACGACTCACGGActcgagGARGTGCAGCTGGTGGAG (Seq. ID No. 41) JMX26VH3b GTCACTCACCAACTCACGACTCACGCActcgagCAGGTGCAGCTGGTGGAG (Seq. ID No. 42) JMX26VH3c CTCACTCACGAACTCACGACTCACGGActcgagGAGGTGCAGCTGTTGGAG (Seq. ID No. 43) JMX26VH4a GTCACTCACGAACTCACGACTCACGGActcgagCAGCTGCAGCTGCAGGAG (Seq. ID No. 44) JMX26VH4b GTCACTCACGAACTCACCACTCACGGActcgagCAGGTGCAGCTACAGCAG (Seq. ID No. 45) JMX26VH5a GTCACTCACGAACTCACGACTCACGGActcgagGARGTGCAGCTGGTGCAG (Seq. ID No. 46) JMX26VH6a GTCACTCACGAACTCACGACTCACGGActcgagCAGGTACAGCTGCAGCAG (Seq. ID No. 47) JMX26VH7a GTCACTCACG4ACTCACGACTCACGGActcgagCAGGTSCAGCTGGTGCAA (Seq. ID No. 48) - In the foregoing sequences (and throughout this disclosure), R is an equal mixture of A and G, K is an equal mixture of G and T, and S is an equal mixture of C and G.
- Blocking oligos for the constant domain of IgM, IgG, and lambda chains are designed. Essentially a region downstream of that required for cloning the genes is identified, and within that region, a sequence useful for annealing a blocking oligo is determined. For example with IgG heavy chains, a native Apa I restriction site present in the CH1 domain can be used for cloning. Generally, the boundary oligo is located downstream of that native restriction site. Compatible nested oligos are then designed which contained all the elements described previously.
- Once amplified, the lambda light chain genes are cloned as is described above for the kappa light chain genes. Likewise, amplified IgG heavy chain fragments are cloned by Xho I/Apa I into an appropriate expression vector that contains, in frame, the remaining portion of the CH1 constant region. Suitable vectors include pRL5, pRL4, fdtetDOG, PHEN1,and pCANTAB5E. Amplified IgM heavy chain fragments are cloned by Xho I/EcoR I into an appropriate expression vector that contains, in frame, the remaining portion of the CH1 constant region. Like the Apa I present natively in IgG genes, the EcoR I site is native to the IgM CH1 domain. Libraries co-expressing both light chains and heavy chains can be screened or selected for Fabs with the desired binding activity.
- First Strand cDNA Synthesis
- Human peripheral blood lymphocyte (PBL) mRNA was used as the original template to generate the first strand cDNA with ThermoScript RT-PCR System (Invitrogen Life Technologies). In addition to oligo dT primer, a phosphoramidate oligonucleotide (synthesized by Annovis Inc. Aston, Pa.) was also included in the reverse transcription reaction. The phosphoramidate oligonucleotide serves as a boundary for reverse transcriptase. The first strand cDNA synthesis was terminated at the location where the phosphoramidate oligonucleotide anneals with the mRNA. The phosphoramidate oligonucleotide, PN-1, was designed to anneal with the framework I region of immunoglobulin (Ig) heavy chain VH3 genes and PN-VH5 was designed to anneal with the framework I region of all the Ig heavy chain genes. A control for first strand cDNA synthesis was also set up by not including the phosphoramidate blocking oligonucleotide. The first strand cDNA product was purified by QIAquick PCR Purification Kit (QIAGEN).
- Phosphoramidate Framework I Blocking Oligonucleotides for Ig Heavy Chain Genes have the following sequences:
(Seq. ID No. 49) PN-1 5′ GCCTCCCCCAGACTC 3′(Seq. ID No. 50) PN- VH5 5′ GCTCCAGACTGCACCAGCTGCAC(C/T) TCGG 3′ - Examination of the Blocking Efficiency
- The blocking efficiency in first strand cDNA synthesis was examined by PCR reactions using blocking check primers and primer CM1, dNTPs, Advantage-2 DNA polymerase mix (Clontech), the reaction buffer, and the first strand cDNA synthesis product. PCR was performed on a PTC-200 thermal cycler (MJ Research) by heating to 94° C. for 30 seconds and followed by cycles of 94° C. for 15 second, 60° C. for 15 second, and 72° C. for one minute. The blocking check primers were designed to anneal with the leader sequences of IgM heavy chain genes. The sequence of CM1, which hybridizes with the CH1 region of IgM, was 5′
GCTCACACTAGTAGGCAGCTCAGCAATCAC 3′ (Seq. ID No. 51). Blocking was analyzed by gel electrophoresis of the PCR products. With appropriate number of cycles, less PCR product was observed from the reverse transcription reactions containing the blocking oligonucleotides than the one does not contain the blocking oligonucleotides, an indication that termination of first strand cDNA synthesis was provided by the hybridization of the blocking oligonucleotides. - The sequences of the blocking check Primers for Ig heavy chain genes have the following sequences:
H1/ 7blck 5′ C TGG ACC TGG AGG ATC C 3′(Seq. ID No. 52) H1blck2 5′ C TGG ACC TGG AGG GTC T 3′(Seq. ID No. 53) H1blck3 5′ C TGG ATT TGG AGG ATC C 3′(Seq. ID No. 54) H2blck1 5′ GACACACTTTGCTCCACG 3′(Seq. ID No. 55) H2blck2 5′ GAC ACA CTT TGC TAC ACA 3′(Seq. ID No. 56) H3blck 5′ TGGGGCTGAGCTGGGTTT 3′(Seq. ID No. 57) H3blck2 5′ TG GGA CTG AGC TGG ATT T 3′(Seq. ID No. 58) H3blck3 5′ TT GGG CTG AGC TGG ATT T 3′(Seq. ID No. 59) H3blck4 5′ TG GGG CTC CCC TGG GTT T 3′(Seq. ID No. 60) H3blck5 5′ TT GGG CTG AGC TGG CTT T 3′(Seq. ID No. 61) H3blck6 5′ TT GGA CTG AGC TGG GTT T 3′(Seq. ID No. 62) H3blck7 5′ TT TGG CTG AGC TGG GTT T 3′(Seq. ID No. 63) H4blck 5′ AAACACCTGTGGTTCTTC 3′(Seq. ID No. 64) H4blck2 5′ AAG CAC CTG TGG TTT TTC 3′(Seq. ID No. 65) H5blck 5′ GCGTCAACCGCCATCCT 3′(Seq. ID No. 66) H6blck 5′ TCTGTCTCCTTCCTCATC 3′(Seq. ID No. 67) - Second Strand cDNA Synthesis and Nesting Oligonucleotide Extension Reaction:
- The purified first strand cDNA synthesis product was used in a nested oligo extension reaction with a hairpin-containing nesting oligonucleotide, dNTPs, Advantage-2, DNA polymerase mix (Clontech), and the reaction buffer. The extension reaction was performed with a GeneAmp PCR System 9700 thermocyler (PE Applied Biosystems). It was heated to 94° C. for 30 seconds and followed by ten cycles of 94° C. for 15 seconds, appropriate annealing temperature for each nesting oligonucleotide for 15 seconds, ramping the temperature to 90° C. at 10% of the normal ramping rate, and 90° C. for 30 seconds. The resulted heavy chain gene should contain a hairpin structure.
Nesting Oligonucleotides for Ig VH1 Heavy Chain genes had the following sequences: hpVH1-1 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAGGTGCAGCTGGTGCAG (Seq. ID No. 68) TCTGGGGCT GAGGTGAAGAAGCCTG AAG 3′hpVH1-2 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGCAGaTGCAGCTGGTGCAG (Seq. ID No. 69) TCTGGGGCTGAGGTGAAGAAGaCTAAT 3′hpVH1-3 5′CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG ATG CAG CTG GTG CAG TCT (Seq. ID No. 70) GGGCCT GAG GTG AAG AAG CCT ATT 3′hpVH1-4 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCAGCTGGTGCAG (Seq. ID No. 71) TCTGGGGCTGAGGTGAAGAAGCCTGAAG 3′ Nesting Oligonucleotides for Ig VH2 Heavy Chain Genes: hpVH2-1 5′CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG ATC ACC TTG AAG GAG TCT (Seq. ID No. 72) GGT CCT ACG CTG GTG AAA CCC ACATAA 3′hpVH2-2 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG GTC ACC TTG AAG GAG TCT (Seq. ID No. 73) GGT CCT GYG CTG GTG AAA CCC AC TAA 3′ Y: C/TNesting Oligonucleotides for Ig VH3 Heavy Chain Genes: hpVH3A1 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG GAG TCT (Seq. ID No. 74) GGG GGA GGC TTG GT(C/A) CAG CCT GGGAAA 3′hpVH3A2 5′CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCAGCTGGTGGAGTCTGGG (Seq. ID No. 75) GGAGGC(T/C)TGGT(A/C) AAGCCTGGGAAA 3′hpVH3A3 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCAGCTGGTGGAGT (Seq. ID No. 76) CTGGGGAGGTGTGGTACGGCCTGGGAAA 3′ hpVH3A4 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGGAGGTGCAGCTGGTGGAGA (Seq. ID No. 77) CTGGAGGAGGCTTGATCCAGCCTGGGAAG 3′ hpVH3A5 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCAGCTGGTGGAGT (Seq. ID No. 78) CTGGGGGAGTCGTGGTACAGCCTGGGAAA 3′ hpVH3A6 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCAGCTGGTGGAGT CT (Seq. ID No. 79) CGGGGAGTCTTGGTACAGCCTGGGAAA 3′ hpVH3A7 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG GA G TCT (Seq. ID No. 80) GGG GGA GGC TTG GTA CAG CCT GGCAAA 3′hpVH3A8 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG GA G TCT (Seq. ID No. 81) GGG GGA GGC TTG GTC CAG CCT GGAAAA 3′hpVH3A9 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG GA G TCT (Seq. ID No. 82) GGG GGA GGC TTA GTT CAG CCT GGGAAA 3′hpVH3A10 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG GA G TCT (Seq. ID No. 83) GGG GGA GGC TTG GTA CAG CCA GGGAAA 3′ots-hp- VH3b 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGCAGGTGCAGCTGGTGGAGT (Seq. ID No. 84) hp- VH3B2 5′ CTCGAGGGCCCGCGAAAGCGGGCCGTCGAGCAGGTGCAGCTGGTGGAGT (Seq. ID No. 85) CTGGGGGAGGCTTGGTCAAGCCTGGAAAG 3′ hpVH3C 5′ CTCGAGGGCCCGCGAAAGCGGGCGCTGGAG GAG GTG GAG CTGTTG GA G TCT (Seq. ID No. 86) GGG GGA GGCTTG GTA GAG CCT GGGAAA 3′Nesting Oligonucleotides for Ig VH4 Heavy Chain Genes: hpVH4-1 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG STG GAG CTG, GAG GA G TCG (Seq. ID No. 87) GGC CGA GGA CTG GTG AAG CCT T AAA 3′ S: C/GhpVH4-2 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG CTG GAG CTG CAG GAG TCG (Seq. ID No. 88) GGC TCA GGA CTG GTG AAG CCT T AAA 3′hpVH4-3 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG AG GTG CAG CTG CAGCAG TGG (Seq. ID No. 89) GGC GCA GGA CTGTTG AAG CCT T AAT 3′Nesting Oligonucleotides for Ig VH5 Heavy Chain Genes: othpVH52 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCAGCTGGTGCAGT CT (Seq. ID No. 90) GGAGCAGAGGTGAAAAAGCCCGGGGAAAA 3′ Nesting Oligonucleotides for Ig VH6 Heavy Chain Genes: hpVH6 5′ +E,usn CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAGGTA GAG CTG GAG GAG TCA (Seq. ID No. 91) GGT CCA GGA CTG GTG AAG CCC AAA 3′Nesting Oligonucleotides for Ig VH7 Heavy Chain Genes: hpVH7 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG GAG CTG GTG CAA TCT (Seq. ID No. 92) GGG TCT GAG TTG AAG AAG CCT ATA 3′Additional Ig Heavy Chain Nesting Oligonucleotides: hpVH 3kb1 5′CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCGACTGGTGGAG (Seq. ID No. 93) TCTGGGGGAGACTTGGTAGAACCGGGGAAG 3′ hpVH 3kb2 5′CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGATGCAACTGGTGGAG (Seq. ID No. 94) TCTGGGGGAGCCTTCGTCCAGCCGGGGAAG 3′ - Single Primer Amplification of IgM Hairpin-Containing Fd Fragments
- Products from the nesting oligo extension reaction (i.e. the engineered template) were amplified using Advantage-2 DNA polymerase mix (Clontech), the reaction buffer, dNTPs, and a single primer named CM3 primer. The sequence for the CM3 primer, which anneals with the CH1 region of IgM, was:
5′ AGAATTTGACTAGTTGGCAAGAGGCACGTTCTTTTCTTTGTTGCCGT 3′.(Seq. ID No. 95) - The amplification reaction was performed with a GeneAmp PCR System 9700 thermocyler (PE Applied Biosystems). It was initially heated to 94° C. for 30 seconds and followed by thirty to forty cycles of 94° C. for 15 seconds, appropriate annealing temperature for 15 seconds, ramping the temperature to 90° C. at 10% of the normal ramping speed, and at 90° C. for 30 seconds. The amplified product was examined by electrophoresis to be of the expected size, ˜0.7 kb. The amplified fragments were cloned into an expression vector and their sequences were confirmed to be human IgM.
- First Strand cDNA Synthesis
- The same protocol as example 3 is employed using mRNA of PBL from a human donor immunized with hepatitis B surface antigen and the phosphoramidate boundary oligonucleotides designed to anneal with the leader sequence of the Ig heavy chain genes. The phosphoramidate leader boundary oligonucleotides for Ig heavy chain genes have the following sequences:
PNVH31d 5′ CACCTCACACTGGACACCTTT 3′(Seq. ID No. 95) PNVH41d 5′ CTGGGACAGGACCCATCTGGG 3′(Seq. ID No. 96) PNVHL1d 5′ TGGGAGTGGGCACCTGTGG 3′(Seq. ID No. 97) PNVH21d 5′ CTGGGACAAGACCCATGAAG 3′(Seq. ID No. 98) PNVH51d 5′ TCGGAACAGACTCCTTGGAGA 3′(Seq. ID No. 99) PNVH61d 5′ CTGTGACAGGACACCCCATGG 3′(Seq. ID No. 100) - Examination of the Blocking Efficiency
- The blocking efficiency in first strand cDNA synthesis is examined by PCR reactions using dNTPs, Advantage-2 DNA polymerase mix (Clontech), the reaction buffer, the first strand cDNA synthesis product, the blocking check primers in Example 3, and the pooled primer mixture of CG1Z, CG2speI, CG3speI, and CG4SpeI. The sequence of primer CG1Z, which hybridized with the CH1 region of IgG1, is 5′
GCATGTACTAGTTTTGTCACAAGATTTGGG 3′. (Seq. ID No. 101) The sequence of primer CG2speI, which hybridized with the CH1 region of IgG2, is 5′AAGGAAACTAGTTTTGCGCTCAACTGTCTTGTCCACCTT 3′. (Seq. ID No. 102) The sequence of primer CG3speI, which hybridized with the CH1 region of IgG3, is 5′AAGGAAACTAGTGTCACCAAGTGGGGTTTTGAGCTC 3′. (Seq. ID No. 103) The sequence of primer CG4speI, which hybridized with the CH1 region of IgG4, is 5′AAGGAAACTAGTACCATATTTGGACTCAACTCTCTTG 3′. (Seq. ID No. 104) PCR is performed on a PTC-200 thermal cycler (MJ Research) by heating to 94° C. for 30 seconds before the following cycle is run, 94° C. for 15 second, 60° C. for 15 second, and 72° C. for one minute. The PCR products were analyzed by gel electrophoresis. With appropriate number of cycles less PCR products were observed from reverse transcription reactions containing the blocking oligonucleotide than the one does not contain blocking oligonucleotide, an indication that termination of first strand cDNA synthesis was provided by hybridization of the leader boundary oligonucleotides. - Second Strand cDNA Synthesis and Nesting Oligonucleotide Extension Reaction:
- The same protocol as Example 3 is employed with nesting oligonucleotides having the following sequences are used.
- Nesting Oligonucleotide's for Ig Heavy Chain VH3 Genes:
HpH3L1 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGSAGGTGCAGCTGGTGGAG (Seq. ID No. 105) TCYGAAA 3′ where S is an equal mixture of C and G, and Y is an equal mixture of T andC HpH3L2 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCAG CTG TTG GAG TCT (Seq. ID No. 106) GAAT 3′HpH3L3 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG GAG ACT (Seq. ID No. 107) GATA 3′HpH3L4 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG GAG TCT (Seq. ID No. 108) CAAA 3′Nesting Oligonucleotides for Ig Heavy Chain VH4 Genes: HpH4L1 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG STG CAG CTG CAG GAG TCG (Seq. ID No. 109) GAAA 3′HpH4L2 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTGGAG CAG CTG CAG CTG CAG GAG TCC (Seq. ID No. 110) AAA 3′HpH4L3 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG GTG CAG CTA CAG CAG TGG (Seq. ID No. 111) GAAA 3′HpH1L1 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG GTB CAG CTK GTG CAG (Seq. ID No. 112) AAA 3′ where B is an equal mixture of C, G and T and K is an equalmixture of G and T HpH1L2 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG SAG GTC CAG CTG GTA CAG AAA 3′(Seq. ID No. 113) where S is an equal mixture of C and G HpH1L3 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG ATG CAG CTG GTG CAG (Seq. ID No. 114) AAA 3′HpH1L4 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAA ATG CAG CTG GTG CAG (Seq. ID No. 115) AAA 3′Nesting Oligonucleotides for Ig Heavy Chain VH2 Genes: HpH2L1 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG ATC ACC TTG AAG GAG TCT (Seq. ID No. 116) AAA 3′HpH2L2 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG GTC ACC TTG AAG GAG TCT (Seq. ID No. 117) AAA 3′Nesting Oligonucleotides for Ig Heavy Chain VH5 Genes: HpH5L1 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG CAG AAA 3′(Seq. ID No. 118) HpH5L2 5′ CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAA GTG CAG CTG GTG CAG AAA 3′(Seq. ID No. 119) Nesting Oligonucleotides for Ig Heavy Chain VH6 Genes: HpH6L1 5′CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG GTA CAG CTG CAG CAG TC (Seq. ID No. 120) AAA 3′Nesting Oligonucleotides for Ig Heavy Chain VH7 Genes: HpH7L1 5′CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG GTG CAG CTG GTG CAA (Seq. ID No. 121) TAAA 3′ - Single Primer Amplification of Human IgG Heavy Chain Fd Hairpin Containing Fragments
- The sample protocol as Example 3 was employed using CG1Z, CG2speI, CG3speI, or CG4SpeI as the primer.
- Cloning of Amplified IgG Heavy Chain Fd Fragments into a Phage Display Vector
- The amplified IgG heavy chain fd hairpin fragments are analyzed by gel electrophoresis. The ˜0.7 kb fragment is separated from the primers by cutting out the gel slice and the DNA was collected by electroelution. The eluted DNA was precipitated by ethanol and resuspended in water. It is digested with restriction enzymes XhoI and SpeI and purified by the QIAquick PCR Purification Kit (QIAGEN). The purified XhoI-SpeI fragment is ligated into a suitable plasmid into which the light chain kappa genes amplified from the same donor had previously been cloned. The ligated reaction was transformed intoE. coli XL-1 Blue strain {F′ proA+B+ lac1q Δ (lacZ) M15 Tn10/recA1 end A1 gyrA96 thi-1 hsdR17 supE44 relA1 lac} by electroporation.
- Selection of Human IgG Antibodies that Bind with the Hepatitis B Surface Antigen
- The XL-1 Blue cells electroporated with the ligation reaction of the phagemid vector and the heavy chain Fd fragments were grown in SOC medium at 37° C. with shaking for one hour. SOC medium is 20 mM glucose in SB medium which contains 1% MOPS hemisodium salt, 3% Bacto Tryptone, and 2% Bacto Yeast Extract. Cells transformed with the plasmid were selected by adding carbenicillin to the culture and they were grown for two hours before infected with a helper phage, VCSM13. After two hours XL-1 Blue cells infected with the helper phage were selected by adding Kanamycin to the culture and the infected cells were amplified overnight by growing at 37° C. with shaking. The next morning the amplified phages were harvested by precipitating with polyethylene glycol (PEG) from the culture supernatant. The PEG precipitated phages were collected by centrifugation. They were resuspended in 1% bovine serum albumin (BSA) in TBS buffer and used in panning for selecting human IgG antibodies that bind with the hepatitis B surface antigen. The resuspended phages were bound with the hepatitis B surface antigen immobilized on the ELISA plate (Costar). The unbound phages were washed off with a washing buffer (0.5
% Tween 20 in PBS) and the bound phages were eluted off the plate with a phage elution buffer (0.1M HCl/glycine, pH 2.2, 1 mg/ml BSA) and neutralized with a neutralization buffer (2M Tris Base). The eluted phages were infected with E. coli ER strain {F′ proA+B+ lac1q Δ (lacZ) M15/fhuA2 (ton A) Δ (lac-proAB) supE thi-1 Δ (hsdMS-mcrB)5}, followed by infection with VCSM13 helper phage. The panning procedure for selecting antibodies bound to hepatitis B surface antigen were repeated three more times. - ELISA Screening of Antibody Clones that Bind with the Hepatitis B Surface Antigen
- Phages eluted at the fourth round of panning were infected withE. coli Top10F′ strain {F′ lac1q, Tn10 (TetR mcrA Δ (mrr-hsdRMS-mcrBC) Φ8(lacZ Δm15 Δlacx74 deoR recA1 araD13 Δ(ara-leu)7697 galU galK [sL(StrR) endA1 nupG) and plated on LB-agar plates containing carbenicilin and tetracycline. Individual clones were picked from the plates and grown overnight in SB medium containing carbenicilin and tetracycline. The IgG Fab fragment will be secreted into the culture supernatant. The next morning cells were removed from these cultures by centrifugation and the culture supernatant was screened in ELISA assay for binding to hepatitis B surface antigen immobilized on the ELISA plates. To reduce false positives the ELISA plates were pre-blocked with BSA before binding with the Fab fragments in culture supernatant. The non-binding Fab fragments were washed off by a washing solution (0.05
% Tween 20 in PBS). Following the wash, plates were incubated with anti-human IgG (Fab′)2 conjugated with alkaline phosphatase (Pierce) which reacts with p-Nitrophenyl phosphate (Sigma), a chromogenic substrate that shows absorbance at OD405. Positive binding clones were identified by a plate reader (Bio RAD Model 1575) with light absorbance at OD405. Among the ninety-four clones screened there were twenty-eight positive clones. - Characterization of the Hepatitis B Surface Antigen Binding Clones
- The IgG heavy chain genes of positive clones from ELISA screening were characterized by DNA sequencing. Plasmid DNA was extracted from the positive clones and sequenced using primers leadVHpAX, NdP, or SeqGZ (Retrogen, San Diego, Calif.). The sequencing primers have the following sequences:
VBVH3A 5′ GAGCCGCACGAGCCCCTCGAGGARGTGCAGCTGGTGGAG 3′(Seq. ID No. 122) VBVH 3B 5′ GAGCCGCACGAGCCCCTCGAGGAGGTGCAGCTGGTGGAG 3′(Seq. ID No. 123) VBVH 3C 5′ GAGCCGCACGAGCCCCTCGAGGAGGTGCAGCTGTTGGAG 3′(Seq. ID No. 124) VBVH 4A 5′ GAGCCGCACGAGCCCCTCGAGCAG(CG) TGCAGCTGCAGGAG 3′(Seq. ID No. 125) VBVH 4B 5′ GAGCCGCACGAGCCCCTCGAGCAGGTGCAGCTACAGCAG 3′(Seq. ID No. 126) LeadVHPAX 5′ GCGGCGCAGCCGGCGATGGCG 3′(Seq. ID No. 127) NdP 5′ AGCGTAGTCCGGAACGTCGTACGG (Seq. ID No. 128) SeqGZ 5′ GAAGTAGTCCTTGACCAG 3′(Seq. ID No. 129) - The sequences of the variable region of these IgG heavy chain genes from nineteen positive clones are shown in FIG. 5. The great diversity of these IgG heavy chain genes shows this method can efficiently amplify the repertoire of human IgG heavy chain genes from immunized donors.
- First Strand cDNA Synthesis
- The same protocol as example 3 is employed using the phosphoramidate boundary oligonucleotides designed to hybridize with the leader sequence of the kappa light chain genes. The phosphoramidate leader boundary oligonucleotides for kappa light chain genes have the following sequences:
(Seq. ID No. 130) PNK11d: 5′ T GTC ACA TCT GGC ACC TGG 3′(Seq. ID No. 131) PNK21d: 5′ TC CCC ACT GGA TCC AGG GAC 3′(Seq. ID No. 132) PNK31d: 5′ C TCC GGT GGT ATC TGG GAG 3′(Seq. ID No. 133) PNK41d: 5′ TC CCC GTA GGC ACC AGA GA 3′(Seq. ID No. 134) PNK51d: 5′ TC TGC CCT GGT AT C AGA GAT 3′(Seq. ID No. 135) PNK61d: 5′ C ACC CCT GGA GGC TGG AAC 3′ - Examination of the Blocking Efficiency
- The blocking efficiency in first Strand cDNA Synthesis was examined by PCR reactions using blocking check primers and primer CK1DX2, dNTPs, Advantage-2 DNA polymerase mix (Clontech), the reaction buffer, and the first strand cDNA synthesis product. PCR was performed on a PTC-200 thermal cycler (MJ Research) by heating to 94° C. for 30 seconds and followed by cycles of 94° C. for 15 second, 60° C. for 15 second, and 72° C. for one minute. The blocking check primers were designed to anneal with the leader sequences of kappa light chain genes. The sequence of CK1DX2, which hybridizes with the constant region of Kappa light chain, was 5′
AGACAGTGAGCGCCGTCTAGAATTAACACTCTCCCCTGTTGAAGCTCTTTGTGACGGGCGAACTCAG 3′. (Seq. ID No. 136) Blocking was analyzed by gel electrophoresis of the PCR products. With appropriate number of cycles less PCR products was observed from reverse transcription reactions containing the blocking oligonucleotide than one that does not contain blocking oligonucleotide, an indication that termination of first strand cDNA synthesis was provided by hybridization of the leader boundary oligonucleotides. - Blocking Check Primers for Kappa Light Chain Genes have the following sequences:
K1b1ck: 5′ CTCCGAGGTGCCAGATGT 3′(Seq. ID No. 137) K1/2b1ck2: 5′ GCT CAG CTC CTG GGG CT 3′(Seq. ID No. 138) K2b1ck: 5′ GTCCCTGGATCCAGTGAG 3′(Seq. ID No. 139) K3b1ck: 5′ CTCCCAGATACCACCGGA 3′(Seq. ID No. 140) K3b1ck2: 5′ GCG CAG CTT CTC TTC CT 3′(Seq. ID No. 141) K3b1ck3: 5′ CAC AGC TTC TTC TTC CTC 3′(Seq. ID No. 142) K4b1ck: 5′ ATCTCTGGTGCCTACGGG 3′(Seq. ID No. 143) KSb1ck: 5′ ATCTCTGATACCAGGGCA 3′(Seq. ID No. 144) K6b1ck: 5′ GTTCCAGCCTCCAGGGGT 3′(Seq. ID No. 145) - Second Strand cDNA Synthesis and Nesting Oligonucleotide Extension Reaction:
- The same protocol as Example 3 is employed using nesting oligonucleotides having the following sequences:
Nesting oligonucleoties for Light Chain Kappa Vk1: HpK1L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GMC ATC CAG ATG ACC CAG TCT (Seq. ID No. 146) CCTAA 3′ wherein M is an equal mixture of A andC HpK1L2 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC AAC ATC CAG ATGACC CAG TCT (Seq. ID No. 147) CC TAA 3′HpK1L3 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GMC ATC CAG TTG ACC CAG TCT (Seq. ID No. 148) CC TAA 3′ whcrein M is an equal mixture of A andC HpK1L4 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GCC ATC CGG ATG ACC CAG TCT (Seq. ID No. 149) CCTAT 3′HpK1L5 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GTC ATC TGG ATG ACC GAG TCT (Seq. ID No. 150) CCTAT 3′Nesting oligonucleotides for Light Chain Kappa Vk2: HpK2L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAT ATT GIG ATG ACC CAG ACT (Seq. ID No. 151) CTTA 3′HpK2L2 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAT GTT GTGATG ACT CAG TCT (Seq. ID No. 152) CC TAA 3′HpK2L3 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAT ATT GIG AIG ACT GAG TCT (Seq. ID No. 153) CCTAA 3′Nesting oligonucleotides for Light Chain Kappa Vk3: HpK3L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAA ATT GTG TTG ACG GAG TCT (Seq. ID No. 154) CCTAA 3′HpK3L2 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAA ATAGTG ATG ACG CAG TCT (Seq. ID No. 155) CCTAA 3′HpK3L3 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAA ATT GTA ATG ACA GAG TCT (Seq. ID No. 156) CCTAA 3′Nesting oligonucleotides for Light Chain Kappa Vk4: HpK4L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAG ATC GTG AIG ACC GAG TCT (Seq. ID No. 157) CCTAT 3′Nesting oligonucleotides for Light Chain Kappa Vk5: HpK5L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAA ACG ACA CTC ACG GAG TCT (Seq. ID No. 158) CCTAA 3′Nesting oligonucleotides for Light Chain Kappa Vk6: HpK6L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAA ATT GTG CTG ACT CAG TCT (Seq. ID No. 159) CCTAT 3′ - Single Primer Amplification of Kappa Hairpin Fragments
- The same protocol as Example 3 is employed using CK1DX2 as the primer.
- First Strand cDNA Synthesis
- The same protocol as example 3 is employed using the following phosphoramidate boundary oligonucleotides designed to hybridize with the leader sequence of the lambda light chain genes. The phosphoramidate boundary oligonucleotides for lambda light chain genes have the sequences:
PNL11d: 5′ CTG GGC CCA GGA CCC TGT GC 3′(Seq. ID No. 160) PNL21d: 5′ CTG GGC CCA GGA CCC TGT 3′.(Seq. ID No. 161) PNL31d: 5′ GA GGC CAC AGA GCC TGT GCA GAG AGT GAG 3′(Seq. ID No. 162) PNL41d1: 5′ CAG AGC ACA GAG ACC TGT GGA3′ (Seq. ID No. 163) PNL41d2: 5′ CTG GGA GAG AGA CCC TGT CCA3′ (Seq. ID No. 164) PNL51d1: 5′ CTG GGA GAG GGA ACC TGT GCA3′ (Seq. ID No. 165) PNL61d1: 5′ ATT GGC CCA AGA ACC TGT GCA3′ (Seq. ID No. 166) PNL71d1: 5′ CTG AGA ATT GGA CCC TGG GCA3′ (Seq. ID No. 167) PNL81d1: 5′ CTG AGA ATC CAC TCC TGA TCC3′ (Seq. ID No. 168) PNL91d1: 5′ CTG GGA GAG GGA CCC TGT 0A03′ (Seq. ID No. 169) PNL101d1: 5′ CTG GAC CAC TGA CAC TGC AGA3′ (Seq. ID No. 170) - Examination of the Blocking Efficiency
- The same protocol as example 3 is employed using the following blocking check primers and primer CL2DX2, dNTPs, Advantage-2 DNA polymerase mix (Clontech), the reaction buffer, and the first strand cDNA synthesis product. The blocking check primers have the following sequences:
L1b1ck: 5′ CAC TGY GCA GGG TCC TGG 3′(Seq. ID No. 171) L2b1ck: 5′ CAG GGC ACA GGG TCC TGG 3′(Seq. ID No. 172) L3b1ck1: 5′ TAC TGC ACA GGA TCC GTG 3′(Seq. ID No. 173) L3b1ck2: 5′ CAC TTT ACA GGT TCT GTG 3′(Seq. ID No. 174) L3b1ck3: 5′ TTC TGC ACA GTC TCT GAG 3′(Seq. ID No. 175) L3b1ck4: 5′ CTC TGC ACA GGC TCT GAG 3′(Seq. ID No. 176) L3b1ck5: 5′ CTT TGC TCA GGT TCT GTG 3′(Seq. ID No. 177) L3b1ck6: 5′ CAC TGC ACA GGC TCT GTG 3′(Seq. ID No. 178) L3b1ck7: 5′ CTC TAC ACA GGC TCT ATT 3′(Seq~ ID No. 179) L3b1ck7: 5′ CTC TGC ACA GTC TCT GTG 3′(Seq. ID No. 180) L4b1ck1: 5′ TTC TCC ACA GGT CTC TGT 3′(Seq. ID No. 181) L4b1ck2: 5′ CAC TGG ACA GGG TCT CTC 3′(Seq. ID No. 182) L5b1ck1: 5′ CAC TGC ACA GGT TCC CTC 3′(Seq. ID No. 183) L6b1ck: 5′ CAC TGC ACA GGT TCT TGG 3′(Seq. ID No. 184) L7b1ck: 5′ TGC TGC CCA GGG TCC AAT 3′(Seq. ID No. 185) L8b1ck: 5′ TAT GGA TCA GGA GTG GAT 3′(Seq. ID No. 186) L9b1ck: 5′ CTC CTC ACA GGG TCC CTC 3′(Seq. ID No. 187) L10b1ck: 5′ CAC TCT GCA GTG TCA GTG 3′(Seq. ID No. 188) - The sequence of CL2DX2, which hybridizes with the CL region of Lambda genes, has this sequence: 5′
AGACAGTGACGCCGTCTA GAATTATGAACATTCTGTAGG 3′ (Seq. ID No. 189). - Second Strand cDNA Synthesis and Nesting Oligonucleotide Extension Reaction:
- The same protocol as Example 3 is employed using the nesting oligonucleotides having the following sequences:
Nesting oligpnucleotides for Lambda Light Chain VL1: HpL1L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG TCT GTG CTG ACT GAG CCA (Seq. ID No. 190) CCAAA 3′HpL1L2 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAG TCT GTG YTG ACG GAG CCG (Seq. ID No. 191) CCAAA3′ Nesting oligonucleotides for Lambda Light Chain VL2: 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG TCT GCC CTG ACT CAG CCT (Seq. ID No. 192) SAAA3′ Nesting oligonucicotides for Lambda Light Chain VL3: HpL3L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC TCC TAT GAG CTG ACT CAG CCA (Seq. ID No. 193) CYAAA3′ HpL3L2 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC TCC TAT GAG CTG ACA CAG CYA (Seq. ID No. 194) CCAAT 3′HpL3L3 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC T CT TCT GAG CTG ACT CAG GAC (Seq. ID No. 195) CCAAA 3′HpL3L4 5′ GAGCTCGGCCCGCGAXAGCGGGCCGAGCTC TCC TAT GTG CTG ACT CAG CCA (Seq. ID No. 196) CCAAA3′ HpL3L5 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTCTCC TAT GAG CTG ATG CAG CCA (Seq. ID No. 197) CCAAA 3′HpL3L6 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC TCC TAT GAG CTG ACA CAG CCA (Seq. ID No. 198) TCAAA3′ Nesting oligonucleotides for Lambda Light Chain VL4: HpL4L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CTG CCT GTG CTG ACT CAG CCC (Seq. ID No. 199) CCAAA3′ HpL4L2 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG CCT GTG CTG ACT CAA TCA (Seq. ID No. 200) TCAAA3′ HpL4L3 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG CTT GTG CTG ACT CAA TCG (Seq. ID No. 201) CCAAA3′ Nesting oligonucleotides for Lambda Light Chain VL5: HpL5L1 5e. 5b 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG CCT GTG CTG ACT CAG CCA (Seq. ID No. 202) YCAAA3 ′ HpLSL2 5c 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG GCT GTG CTG ACT CAG CCG (Seq. ID No. 203) GCAAA3′ Nesting oligonuele6tides for Lambda Light Chain VL6: HpL6L1 6a 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC AAT TTT ATG CTG ACT CAG CCC (Seq. ID No. 204) CAAAA3′ Nesting oligonucleotides for Lambda Light Chain VL7 and VL8: HpL7/ 8L1 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG ACT GTG GTG ACY CAG GAG (Seq. ID No. 205) CCAAA3′ HpL7L2 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC G CAG GCT GTG GTG ACT CAG (Seq. ID No. 206) GAG CCAAA3′ Nesting oligonucleotides for Lambda Light Chain VL9; HpL9L 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG CCT GTG CTG ACT CAG CCA (Seq. ID No. 207) CCAAA3′ Nesting oligonucleotides for Lambda Light Chain VL10: 5′ GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG GCA GGG CTG ACT CAG CCA (Seq. ID No. 208) CCAAA3′ - Single Primer Amplification of Lambda Hairpin Containing Fragments
- The same protocol as Example 3 is employed using CL2DX2 as the primer.
- First Strand cDNA Synthesis
- The same protocol as example 3 was employed using mRNA of PBL from a human donor immunized with hepatitis B surface antigen as the original template using blocking oligonucleotides that anneal to FR1 of the variable heavy chain.
- Examination of the Blocking Efficiency
- The same protocol as example 4 was employed.
- Second Strand cDNA Synthesis and Nesting Oligonucleotide Extension Reaction:
- The same protocol as Example 3 was employed.
- Single Primer Amplification of Human IgG Heavy Chain Fd Hairpin Containing Fragments
- The sample protocol as Example 4 was employed.
- Cloning of Amplified IgG Heavy Chain Fd Fragments into a Phage Display Vector
- The sample protocol as Example 4 was employed.
- Selection of Human IgG Antibodies that Bind with the Hepatitis B Surface Antigen
- The sample protocol as Example 4 was employed.
- ELISA Screening of Antibody Clones that Bind with the Hepatitis B Surface Antigen
- The sample protocol as Example 4 was employed. Among the ninety-four clones screened eighty clones are positive.
- Characterization of the Hepatitis B Surface Antigen Binding Clones
- The sample protocol as Example 4 was employed. Sequences of the variable regions of the heavy chain genes from fourteen positive clones are listed in FIG. 6. The sequence diversity of these clones and others produced shows this method can efficiently amplify the repertoire of human heavy chain genes from immunized donors.
- It will be understood that various modifications may be made to the embodiments described herein. Therefore, the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. Those skilled in the art will envision other modifications within the scope and spirit of this disclosure.
-
1 231 1 51 DNA artificial sequence primer 1 gtcactcacg aactcacgac tcacggagag ctcracatcc agatgaccca g 51 2 21 DNA artificial sequence blocking oligonucleotide 2 gaactgtggc tgcaccatct g 21 3 93 DNA artificial sequence nested/hairpin oligonucleotide 3 ccttagagtc acgctagcga ttgattgatt gattgattgt ttgtgactct aaggttggcg 60 cgccttcgtt tgatytccac cttggtccnt ngn 93 4 26 DNA artificial sequence primer 4 gtcactcacg aactcacgac tcacgg 26 5 22 DNA artificial sequence primer 5 cacgctagcg attgattgat tg 22 6 45 DNA artificial sequence primer 6 gaggaggagg aggaggaggg cgcgcctgat ytccaccttg gtccc 45 7 51 DNA artificial sequence primer 7 gtcactcacg aactcacgac tcacggagag ctcracatcc agatgaccca g 51 8 51 DNA artificial sequence primer 8 gtcactcacg aactcacgac tcacggagag ctcgmcatcc agttgaccca g 51 9 51 DNA artificial sequence primer 9 gtcactcacg aactcacgac tcacggagag ctcgccatcc rgatgaccca g 51 10 51 DNA artificial sequence primer 10 gtcactcacg aactcacgac tcacggagag ctcgtcatct ggatgaccca g 51 11 51 DNA artificial sequence primer 11 gtcactcacg aactcacgac tcacggagag ctcgatattg tgatgaccca g 51 12 51 DNA artificial sequence primer 12 gtcactcacg aactcacgac tcacggagag ctcgatrttg tgatgactca g 51 13 51 DNA artificial sequence primer 13 gtcactcacg aactcacgac tcacggagag ctcgaaattg tgttgacrca g 51 14 51 DNA artificial sequence primer 14 gtcactcacg aactcacgac tcacggagag ctcgaaatag tgatgacgca g 51 15 51 DNA artificial sequence primer 15 gtcactcacg aactcacgac tcacggagag ctcgaaattg taatgacaca g 51 16 51 DNA artificial sequence primer 16 gtcactcacg aactcacgac tcacggagag ctcgacatcg tgatgaccca g 51 17 51 DNA artificial sequence primer 17 gtcactcacg aactcacgac tcacggagag ctcgaaacga cactcacgca g 51 18 51 DNA artificial sequence primer 18 gtcactcacg aactcacgac tcacggagag ctcgaaattg tgctgactca g 51 19 51 DNA artificial sequence primer 19 gtcactcacg aactcacgac tcacggagag ctcgatgttg tgatgacaca g 51 20 51 DNA artificial sequence primer 20 gtcactcacg aactcacgac tcacggagag ctccagtctg tgctgactca g 51 21 51 DNA artificial sequence primer 21 gtcactcacg aactcacgac tcacggagag ctccagtctg tgytgacgca g 51 22 51 DNA artificial sequence primer 22 gtcactcacg aactcacgac tcacggagag ctccagtctg tcgtgacgca g 51 23 51 DNA artificial sequence primer 23 gtcactcacg aactcacgac tcacggagag ctccagtctg ccctgactca g 51 24 51 DNA artificial sequence primer 24 gtcactcacg aactcacgac tcacggagag ctctcctatg wgctgactca g 51 25 51 DNA artificial sequence primer 25 gtcactcacg aactcacgac tcacggagag ctctcctatg agctgacaca g 51 26 51 DNA artificial sequence primer 26 gtcactcacg aactcacgac tcacggagag ctctcttctg agctgactca g 51 27 51 DNA artificial sequence primer 27 gtcactcacg aactcacgac tcacggagag ctctcctatg agctgatgca g 51 28 51 DNA artificial sequence primer 28 gtcactcacg aactcacgac tcacggagag ctccagcytg tgctgactca a 51 29 51 DNA artificial sequence primer 29 gtcactcacg aactcacgac tcacggagag ctccagsctg tgctgactca g 51 30 51 DNA artificial sequence primer 30 gtcactcacg aactcacgac tcacggagag ctcaatttta tgctgactca g 51 31 51 DNA artificial sequence primer 31 gtcactcacg aactcacgac tcacggagag ctccagrctg tggtgactca g 51 32 51 DNA artificial sequence primer 32 gtcactcacg aactcacgac tcacggagag ctccagactg tggtgaccca g 51 33 51 DNA artificial sequence primer 33 gtcactcacg aactcacgac tcacggagag ctccwgcctg tgctgactca g 51 34 51 DNA artificial sequence primer 34 gtcactcacg aactcacgac tcacggagag ctccaggcag ggctgactca g 51 35 51 DNA artificial sequence primer 35 gtcactcacg aactcacgac tcacggactc gagcaggtkc agctggtgca g 51 36 51 DNA artificial sequence primer 36 gtcactcacg aactcacgac tcacggactc gagcaggtcc agcttgtgca g 51 37 51 DNA artificial sequence primer 37 gtcactcacg aactcacgac tcacggactc gagsaggtcc agctggtaca g 51 38 51 DNA artificial sequence primer 38 gtcactcacg aactcacgac tcacggactc gagcaratgc agctggtgca g 51 39 51 DNA artificial sequence primer 39 gtcactcacg aactcacgac tcacggactc gagcagatca ccttgaagga g 51 40 51 DNA artificial sequence primer 40 gtcactcacg aactcacgac tcacggactc gagcaggtca ccttgargga g 51 41 51 DNA artificial sequence primer 41 gtcactcacg aactcacgac tcacggactc gaggargtgc agctggtgga g 51 42 51 DNA artificial sequence primer 42 gtcactcacg aactcacgac tcacggactc gagcaggtgc agctggtgga g 51 43 51 DNA artificial sequence primer 43 gtcactcacg aactcacgac tcacggactc gaggaggtgc agctgttgga g 51 44 51 DNA artificial sequence primer 44 gtcactcacg aactcacgac tcacggactc gagcagstgc agctgcagga g 51 45 51 DNA artificial sequence primer 45 gtcactcacg aactcacgac tcacggactc gagcaggtgc agctacagca g 51 46 51 DNA artificial sequence primer 46 gtcactcacg aactcacgac tcacggactc gaggargtgc agctggtgca g 51 47 51 DNA artificial sequence primer 47 gtcactcacg aactcacgac tcacggactc gagcaggtac agctgcagca g 51 48 51 DNA artificial sequence primer 48 gtcactcacg aactcacgac tcacggactc gagcaggtsc agctggtgca a 51 49 15 DNA artificial sequence blocking oligonucleotide 49 gcctccccca gactc 15 50 28 DNA artificial sequence blocking oligonucleotide 50 gctccagact gcaccagctg cacntcgg 28 51 30 DNA artificial sequence primer 51 gctcacacta gtaggcagct cagcaatcac 30 52 17 DNA artificial sequence primer 52 ctggacctgg aggatcc 17 53 17 DNA artificial sequence primer 53 ctggacctgg agggtct 17 54 17 DNA artificial sequence primer 54 ctggatttgg aggatcc 17 55 18 DNA artificial sequence primer 55 gacacacttt gctccacg 18 56 18 DNA artificial sequence primer 56 gacacacttt gctacaca 18 57 18 DNA artificial sequence primer 57 tggggctgag ctgggttt 18 58 18 DNA artificial sequence primer 58 tgggactgag ctggattt 18 59 18 DNA artificial sequence primer 59 ttgggctgag ctggattt 18 60 18 DNA artificial sequence primer 60 tggggctccg ctgggttt 18 61 18 DNA artificial sequence primer 61 ttgggctgag ctggcttt 18 62 18 DNA artificial sequence primer 62 ttggactgag ctgggttt 18 63 18 DNA artificial sequence primer 63 tttggctgag ctgggttt 18 64 18 DNA artificial sequence primer 64 aaacacctgt ggttcttc 18 65 18 DNA artificial sequence primer 65 aagcacctgt ggtttttc 18 66 17 DNA artificial sequence primer 66 gggtcaaccg ccatcct 17 67 18 DNA artificial sequence primer 67 tctgtctcct tcctcatc 18 68 76 DNA artificial sequence nesting oligonucleotide 68 ctcgagggcc cgcgaaagcg ggccctcgag caggtgcagc tggtgcagtc tggggctgag 60 gtgaagaagc ctgaag 76 69 75 DNA artificial sequence nesting oligonucleotide 69 ctcgagggcc cgcgaaagcg ggccctcgag cagatgcagc tggtgcagtc tggggctgag 60 gtgaagaaga ctaat 75 70 75 DNA artificial sequence nesting oligonucleotide 70 ctcgagggcc cgcgaaagcg ggccctcgag cagatgcagc tggtgcagtc tgggcctgag 60 gtgaagaagc ctatt 75 71 76 DNA artificial sequence nesting oligonucleotide 71 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tggtgcagtc tggggctgag 60 gtgaagaagc ctgaag 76 72 78 DNA artificial sequence nesting oligonucleotide 72 ctcgagggcc cgcgaaagcg ggccctcgag cagatcacct tgaaggagtc tggtcctacg 60 ctggtgaaac ccacataa 78 73 77 DNA artificial sequence nesting oligonucleotide 73 ctcgagggcc cgcgaaagcg ggccctcgag caggtcacct tgaaggagtc tggtcctgyg 60 ctggtgaaac ccactaa 77 74 78 DNA artificial sequence nesting oligonucleotide 74 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tggtggagtc tgggggaggc 60 ttggtncagc ctgggaaa 78 75 78 DNA artificial sequence nesting oligonucleotide 75 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tggtggagtc tgggggaggc 60 ntggtnaagc ctgggaaa 78 76 78 DNA artificial sequence nesting oligonucleotide 76 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tggtggagtc tgggggaggt 60 gtggtacggc ctgggaaa 78 77 78 DNA artificial sequence nesting oligonucleotide 77 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tggtggagac tggaggaggc 60 ttgatccagc ctgggaag 78 78 78 DNA artificial sequence nesting oligonucleotide 78 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tggtggagtc tgggggagtc 60 gtggtacagc ctgggaaa 78 79 78 DNA artificial sequence nesting oligonucleotide 79 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tggtggagtc tcggggagtc 60 ttggtacagc ctgggaaa 78 80 78 DNA artificial sequence nesting oligonucleotide 80 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tggtggagtc tgggggaggc 60 ttggtacagc ctggcaaa 78 81 78 DNA artificial sequence nesting oligonucleotide 81 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tggtggagtc tgggggaggc 60 ttggtccagc ctggaaaa 78 82 78 DNA artificial sequence nesting oligonucleotide 82 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tggtggagtc tgggggaggc 60 ttagttcagc ctgggaaa 78 83 78 DNA artificial sequence nesting oligonucleotide 83 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tggtggagtc tgggggaggc 60 ttggtacagc cagggaaa 78 84 78 DNA artificial sequence nesting oligonucleotide 84 ctcgagggcc cgcgaaagcg ggccctcgag caggtgcagc tggtggagtc tgggggaggc 60 gtggtccagc ctgggttt 78 85 78 DNA artificial sequence nesting oligonucleotide 85 ctcgagggcc cgcgaaagcg ggccctcgag caggtgcagc tggtggagtc tgggggaggc 60 ttggtcaagc ctggaaag 78 86 78 DNA artificial sequence nesting oligonucleotide 86 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tgttggagtc tgggggaggc 60 ttggtacagc ctgggaaa 78 87 76 DNA artificial sequence nesting oligonucleotide 87 ctcgagggcc cgcgaaagcg ggccctcgag cagstgcagc tgcaggagtc gggcccagga 60 ctggtgaagc cttaaa 76 88 76 DNA artificial sequence nesting oligonucleotide 88 ctcgagggcc cgcgaaagcg ggccctcgag cagctgcagc tgcaggagtc gggctcagga 60 ctggtgaagc cttaaa 76 89 75 DNA artificial sequence nesting oligonucleotide 89 ctcgagggcc cgcgaaagcg ggccctcgag aggtgcagct gcagcagtgg ggcgcaggac 60 tgttgaagcc ttaat 75 90 80 DNA artificial sequence nesting oligonucleotide 90 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tggtgcagtc tggagcagag 60 gtgaaaaagc ccggggaaaa 80 91 75 DNA artificial sequence nesting oligonucleotide 91 ctcgagggcc cgcgaaagcg ggccctcgag caggtacagc tgcagcagtc aggtccagga 60 ctggtgaagc ccaaa 75 92 75 DNA artificial sequence nesting oligonucleotide 92 ctcgagggcc cgcgaaagcg ggccctcgag caggtgcagc tggtgcaatc tgggtctgag 60 ttgaagaagc ctata 75 93 78 DNA artificial sequence nesting oligonucleotide 93 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcgac tggtggagtc tgggggagac 60 ttggtagaac cggggaag 78 94 78 DNA artificial sequence nesting oligonucleotide 94 ctcgagggcc cgcgaaagcg ggccctcgag gagatgcaac tggtggagtc tgggggagcc 60 ttcgtccagc cggggaag 78 95 21 DNA artificial sequence boundary oligonucleotide 95 cacctcacac tggacacctt t 21 96 21 DNA artificial sequence boundary oligonucleotide 96 ctgggacagg acccatctgg g 21 97 19 DNA artificial sequence boundary oligonucleotide 97 tgggagtggg cacctgtgg 19 98 20 DNA artificial sequence boundary oligonucleotide 98 ctgggacaag acccatgaag 20 99 21 DNA artificial sequence boundary oligonucleotide 99 tcggaacaga ctccttggag a 21 100 21 DNA artificial sequence boundary oligonucleotide 100 ctgtgacagg acaccccatg g 21 101 30 DNA artificial sequence primer 101 gcatgtacta gttttgtcac aagatttggg 30 102 39 DNA artificial sequence primer 102 aaggaaacta gttttgcgct caactgtctt gtccacctt 39 103 36 DNA artificial sequence primer 103 aaggaaacta gtgtcaccaa gtggggtttt gagctc 36 104 37 DNA artificial sequence primer 104 aaggaaacta gtaccatatt tggactcaac tctcttg 37 105 55 DNA artificial sequence nesting oligonucleotide 105 ctcgagggcc cgcgaaagcg ggccctcgag saggtgcagc tggtggagtc ygaaa 55 106 55 DNA artificial sequence nesting oligonucleotide 106 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tgttggagtc tgaat 55 107 55 DNA artificial sequence nesting oligonucleotide 107 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tggtggagac tgata 55 108 55 DNA artificial sequence nesting oligonucleotide 108 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tggtggagtc tcaaa 55 109 55 DNA artificial sequence nesting oligonucleotide 109 ctcgagggcc cgcgaaagcg ggccctcgag cagstgcagc tgcaggagtc ggaaa 55 110 54 DNA artificial sequence nesting oligonucleotide 110 ctcgagggcc cgcgaaagcg ggccctcgag cagctgcagc tgcaggagtc caaa 54 111 55 DNA artificial sequence nesting oligonucleotide 111 ctcgagggcc cgcgaaagcg ggccctcgag caggtgcagc tacagcagtg ggaaa 55 112 51 DNA artificial sequence nesting oligonucleotide 112 ctcgagggcc cgcgaaagcg ggccctcgag caggtbcagc tkgtgcagaa a 51 113 51 DNA artificial sequence nesting oligonucleotide 113 ctcgagggcc cgcgaaagcg ggccctcgag saggtccagc tggtacagaa a 51 114 51 DNA artificial sequence nesting oligonucleotide 114 ctcgagggcc cgcgaaagcg ggccctcgag cagatgcagc tggtgcagaa a 51 115 51 DNA artificial sequence nesting oligonucleotide 115 ctcgagggcc cgcgaaagcg ggccctcgag caaatgcagc tggtgcagaa a 51 116 54 DNA artificial sequence nesting oligonucleotide 116 ctcgagggcc cgcgaaagcg ggccctcgag cagatcacct tgaaggagtc taaa 54 117 54 DNA artificial sequence nesting oligonucleotide 117 ctcgagggcc cgcgaaagcg ggccctcgag caggtcacct tgaaggagtc taaa 54 118 51 DNA artificial sequence nesting oligonucleotide 118 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc tggtgcagaa a 51 119 51 DNA artificial sequence nesting oligonucleotide 119 ctcgagggcc cgcgaaagcg ggccctcgag gaagtgcagc tggtgcagaa a 51 120 53 DNA artificial sequence nesting oligonucleotide 120 ctcgagggcc cgcgaaagcg ggccctcgag caggtacagc tgcagcagtc aaa 53 121 52 DNA artificial sequence nesting oligonucleotide 121 ctcgagggcc cgcgaaagcg ggccctcgag caggtgcagc tggtgcaata aa 52 122 39 DNA artificial sequence primer 122 gagccgcacg agcccctcga ggargtgcag ctggtggag 39 123 39 DNA artificial sequence primer 123 gagccgcacg agcccctcga ggaggtgcag ctggtggag 39 124 39 DNA artificial sequence primer 124 gagccgcacg agcccctcga ggaggtgcag ctgttggag 39 125 39 DNA artificial sequence primer 125 gagccgcacg agcccctcga gcagntgcag ctgcaggag 39 126 39 DNA artificial sequence primer 126 gagccgcacg agcccctcga gcaggtgcag ctacagcag 39 127 21 DNA artificial sequence primer 127 gcggcgcagc cggcgatggc g 21 128 24 DNA artificial sequence primer 128 agcgtagtcc ggaacgtcgt acgg 24 129 18 DNA artificial sequence primer 129 gaagtagtcc ttgaccag 18 130 19 DNA artificial sequence boundary oligonucleotide 130 tgtcacatct ggcacctgg 19 131 20 DNA artificial sequence boundary oligonucleotide 131 tccccactgg atccagggac 20 132 19 DNA artificial sequence boundary oligonucleotide 132 ctccggtggt atctgggag 19 133 19 DNA artificial sequence boundary oligonucleotide 133 tccccgtagg caccagaga 19 134 20 DNA artificial sequence boundary oligonucleotide 134 tctgccctgg tatcagagat 20 135 19 DNA artificial sequence boundary oligonucleotide 135 cacccctgga ggctggaac 19 136 67 DNA artificial sequence primer 136 agacagtgag cgccgtctag aattaacact ctcccctgtt gaagctcttt gtgacgggcg 60 aactcag 67 137 18 DNA artificial sequence primer 137 ctccgaggtg ccagatgt 18 138 17 DNA artificial sequence primer 138 gctcagctcc tggggct 17 139 18 DNA artificial sequence primer 139 gtccctggat ccagtgag 18 140 18 DNA artificial sequence primer 140 ctcccagata ccaccgga 18 141 17 DNA artificial sequence primer 141 gcgcagcttc tcttcct 17 142 18 DNA artificial sequence primer 142 cacagcttct tcttcctc 18 143 18 DNA artificial sequence primer 143 atctctggtg cctacggg 18 144 18 DNA artificial sequence primer 144 atctctgata ccagggca 18 145 18 DNA artificial sequence primer 145 gttccagcct ccaggggt 18 146 56 DNA artificial sequence nesting oligonucleotide 146 gagctcggcc cgcgaaagcg ggccgagctc gmcatccaga tgacccagtc tcctaa 56 147 56 DNA artificial sequence nesting oligonucleotide 147 gagctcggcc cgcgaaagcg ggccgagctc aacatccaga tgacccagtc tcctaa 56 148 56 DNA artificial sequence nesting oligonucleotide 148 gagctcggcc cgcgaaagcg ggccgagctc gmcatccagt tgacccagtc tcctaa 56 149 56 DNA artificial sequence nesting oligonucleotide 149 gagctcggcc cgcgaaagcg ggccgagctc gccatccgga tgacccagtc tcctat 56 150 56 DNA artificial sequence nesting oligonucleotide 150 gagctcggcc cgcgaaagcg ggccgagctc gtcatctgga tgacccagtc tcctat 56 151 55 DNA artificial sequence nesting oligonucleotide 151 gagctcggcc cgcgaaagcg ggccgagctc gatattgtga tgacccagac tctta 55 152 56 DNA artificial sequence nesting oligonucleotide 152 gagctcggcc cgcgaaagcg ggccgagctc gatgttgtga tgactcagtc tcctaa 56 153 56 DNA artificial sequence nesting oligonucleotide 153 gagctcggcc cgcgaaagcg ggccgagctc gatattgtga tgactcagtc tcctaa 56 154 56 DNA artificial sequence nesting oligonucleotide 154 gagctcggcc cgcgaaagcg ggccgagctc gaaattgtgt tgacgcagtc tcctaa 56 155 56 DNA artificial sequence nesting oligonucleotide 155 gagctcggcc cgcgaaagcg ggccgagctc gaaatagtga tgacgcagtc tcctaa 56 156 56 DNA artificial sequence nesting oligonucleotide 156 gagctcggcc cgcgaaagcg ggccgagctc gaaattgtaa tgacacagtc tcctaa 56 157 56 DNA artificial sequence nesting oligonucleotide 157 gagctcggcc cgcgaaagcg ggccgagctc gacatcgtga tgacccagtc tcctat 56 158 56 DNA artificial sequence nesting oligonucleotide 158 gagctcggcc cgcgaaagcg ggccgagctc gaaacgacac tcacgcagtc tcctaa 56 159 56 DNA artificial sequence nesting oligonucleotide 159 gagctcggcc cgcgaaagcg ggccgagctc gaaattgtgc tgactcagtc tcctat 56 160 20 DNA artificial sequence boundary oligonucleotide 160 ctgggcccag gaccctgtgc 20 161 18 DNA artificial sequence boundary oligonucleotide 161 ctgggcccag gaccctgt 18 162 29 DNA artificial sequence boundary oligonucleotide 162 gaggccacag agcctgtgca gagagtgag 29 163 21 DNA artificial sequence boundary oligonucleotide 163 cagagcacag agacctgtgg a 21 164 21 DNA artificial sequence boundary oligonucleotide 164 ctgggagaga gaccctgtcc a 21 165 21 DNA artificial sequence boundary oligonucleotide 165 ctgggagagg gaacctgtgc a 21 166 21 DNA artificial sequence boundary oligonucleotide 166 attggcccaa gaacctgtgc a 21 167 21 DNA artificial sequence boundary oligonucleotide 167 ctgagaattg gaccctgggc a 21 168 21 DNA artificial sequence boundary oligonucleotide 168 ctgagaatcc actcctgatc c 21 169 21 DNA artificial sequence boundary oligonucleotide 169 ctgggagagg gaccctgtga g 21 170 21 DNA artificial sequence boundary oligonucleotide 170 ctggaccact gacactgcag a 21 171 18 DNA artificial sequence primer 171 cactgygcag ggtcctgg 18 172 18 DNA artificial sequence primer 172 cagggcacag ggtcctgg 18 173 18 DNA artificial sequence primer 173 tactgcacag gatccgtg 18 174 18 DNA artificial sequence primer 174 cactttacag gttctgtg 18 175 18 DNA artificial sequence primer 175 ttctgcacag tctctgag 18 176 18 DNA artificial sequence primer 176 ctctgcacag gctctgag 18 177 18 DNA artificial sequence primer 177 ctttgctcag gttctgtg 18 178 18 DNA artificial sequence primer 178 cactgcacag gctctgtg 18 179 18 DNA artificial sequence primer 179 ctctacacag gctctatt 18 180 18 DNA artificial sequence primer 180 ctctgcacag tctctgtg 18 181 18 DNA artificial sequence primer 181 ttctccacag gtctctgt 18 182 18 DNA artificial sequence primer 182 cactggacag ggtctctc 18 183 18 DNA artificial sequence primer 183 cactgcacag gttccctc 18 184 18 DNA artificial sequence primer 184 cactgcacag gttcttgg 18 185 18 DNA artificial sequence primer 185 tgctgcccag ggtccaat 18 186 18 DNA artificial sequence primer 186 tatggatcag gagtggat 18 187 18 DNA artificial sequence primer 187 ctcctcacag ggtccctc 18 188 18 DNA artificial sequence primer 188 cactctgcag tgtcagtg 18 189 39 DNA artificial sequence primer 189 agacagtgac gccgtctaga attatgaaca ttctgtagg 39 190 56 DNA artificial sequence nesting oligonucleotide 190 gagctcggcc cgcgaaagcg ggccgagctc cagtctgtgc tgactcagcc accaaa 56 191 56 DNA artificial sequence nesting oligonucleotide 191 gagctcggcc cgcgaaagcg ggccgagctc cagtctgtgy tgacgcagcc gccaaa 56 192 55 DNA artificial sequence nesting oligonucleotide 192 gagctcggcc cgcgaaagcg ggccgagctc cagtctgccc tgactcagcc tsaaa 55 193 56 DNA artificial sequence nesting oligonucleotide 193 gagctcggcc cgcgaaagcg ggccgagctc tcctatgagc tgactcagcc acyaaa 56 194 56 DNA artificial sequence nesting oligonucleotide 194 gagctcggcc cgcgaaagcg ggccgagctc tcctatgagc tgacacagcy accaat 56 195 56 DNA artificial sequence nesting oligonucleotide 195 gagctcggcc cgcgaaagcg ggccgagctc tcttctgagc tgactcagga cccaaa 56 196 56 DNA artificial sequence nesting oligonucleotide 196 gagctcggcc cgcgaaagcg ggccgagctc tcctatgtgc tgactcagcc accaaa 56 197 56 DNA artificial sequence nesting oligonucleotide 197 gagctcggcc cgcgaaagcg ggccgagctc tcctatgagc tgatgcagcc accaaa 56 198 56 DNA artificial sequence nesting oligonucleotide 198 gagctcggcc cgcgaaagcg ggccgagctc tcctatgagc tgacacagcc atcaaa 56 199 56 DNA artificial sequence nesting oligonucleotide 199 gagctcggcc cgcgaaagcg ggccgagctc ctgcctgtgc tgactcagcc cccaaa 56 200 56 DNA artificial sequence nesting oligonucleotide 200 gagctcggcc cgcgaaagcg ggccgagctc cagcctgtgc tgactcaatc atcaaa 56 201 56 DNA artificial sequence nesting oligonucleotide 201 gagctcggcc cgcgaaagcg ggccgagctc cagcttgtgc tgactcaatc gccaaa 56 202 56 DNA artificial sequence nesting oligonucleotide 202 gagctcggcc cgcgaaagcg ggccgagctc cagcctgtgc tgactcagcc aycaaa 56 203 56 DNA artificial sequence nesting oligonucleotide 203 gagctcggcc cgcgaaagcg ggccgagctc caggctgtgc tgactcagcc ggcaaa 56 204 56 DNA artificial sequence nesting oligonucleotide 204 gagctcggcc cgcgaaagcg ggccgagctc aattttatgc tgactcagcc ccaaaa 56 205 56 DNA artificial sequence nesting oligonucleotide 205 gagctcggcc cgcgaaagcg ggccgagctc cagactgtgg tgacycagga gccaaa 56 206 57 DNA artificial sequence nesting oligonucleotide 206 gagctcggcc cgcgaaagcg ggccgagctc gcaggctgtg gtgactcagg agccaaa 57 207 56 DNA artificial sequence nesting oligonucleotide 207 gagctcggcc cgcgaaagcg ggccgagctc cagcctgtgc tgactcagcc accaaa 56 208 56 DNA artificial sequence nesting oligonucleotide 208 gagctcggcc cgcgaaagcg ggccgagctc caggcagggc tgactcagcc accaaa 56 209 115 PRT artificial sequence cloned antibody 209 Glu Ser Asp Gly Ala Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Ala Ser Gly Phe Ile Phe Asp Asp Phe Ala Met His Trp Leu 20 25 30 Arg Gln Val Pro Gly Lys Gly Leu Gln Trp Val Gly Leu Met Ser Trp 35 40 45 Asp Gly Val Ser Ala Tyr Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr 50 55 60 Ile Ser Arg Asp Asn Lys Lys Asn Ala Leu Tyr Leu Gln Met Asn Ser 65 70 75 80 Leu Gly Val Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Asp Met Gly 85 90 95 Gly Gly Leu Arg Phe Pro His Phe Trp Gly Gln Gly Thr Pro Val Thr 100 105 110 Val Ser Ala 115 210 110 PRT artificial sequence cloned antibody 210 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 1 5 10 15 Leu Ser Ser Ser Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly 20 25 30 Leu Glu Phe Val Ala Val Ser Ser Gly Asn Gly Phe Ser Thr Tyr Tyr 35 40 45 Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 50 55 60 Asn Met Val Tyr Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala 65 70 75 80 Lys Tyr His Cys Ala Lys Val Arg Tyr Gly Pro Arg Ser His Phe Phe 85 90 95 Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 211 110 PRT artificial sequence cloned antibody 211 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 1 5 10 15 Leu Ser Ser Ser Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly 20 25 30 Leu Glu Phe Val Ala Val Ser Ser Gly Asn Gly Phe Ser Thr Tyr Tyr 35 40 45 Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 50 55 60 Asn Met Val Tyr Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala 65 70 75 80 Lys Tyr His Cys Ala Lys Val Arg Tyr Gly Pro Arg Ser His Phe Phe 85 90 95 Phe Asp Pro Trp Gly Pro Gly Asn Pro Gly His Arg Leu Leu 100 105 110 212 112 PRT artificial sequence cloned antibody 212 Ala Trp Tyr Ser Arg Gly Ser Pro Cys Leu Ser Cys Ala Ala Ser Gly 1 5 10 15 Phe Thr Leu Ser Ser Ser Ala Met Ser Trp Val Arg Gln Ala Pro Gly 20 25 30 Lys Gly Leu Glu Phe Val Ala Val Ser Ser Gly Asn Gly Phe Ser Thr 35 40 45 Tyr Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 50 55 60 Ser Lys Asn Met Val Tyr Leu Gln Met Asp Ser Leu Arg Ala Glu Asp 65 70 75 80 Thr Ala Lys Tyr His Cys Ala Lys Val Arg Tyr Gly Pro Arg Ser His 85 90 95 Phe Phe Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 213 122 PRT artificial sequence cloned antibody 213 Glu Ser Asp Pro Gly Leu Val Lys Pro Ser Glu Thr Pro Ser Leu Thr 1 5 10 15 Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Thr Met Tyr Phe Trp Gly 20 25 30 Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Ala Ser Ile 35 40 45 Tyr Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Arg Ser Arg Val 50 55 60 Thr Met Ser Val Asp Thr Ser Lys Asn Gln Leu Ser Leu Lys Leu Asn 65 70 75 80 Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Pro Thr 85 90 95 Ile Tyr Tyr Phe Asp Gly Arg Thr Ser Tyr Tyr Pro Gly Glu Ala Ala 100 105 110 Phe Asp Ile Trp Gly Gln Gly Thr Thr Val 115 120 214 121 PRT artificial sequence cloned antibody 214 Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val 1 5 10 15 Ser Gly Gly Ser Ile Ser Asn Ile Met Tyr Phe Trp Gly Trp Ile Arg 20 25 30 Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Ala Ser Ile Tyr Tyr Ser 35 40 45 Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Arg Ser Arg Val Thr Met Ser 50 55 60 Val Asp Thr Ser Lys Asn Gln Leu Ser Leu Lys Leu Asn Ser Val Thr 65 70 75 80 Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Pro Thr Ile Tyr Tyr 85 90 95 Phe Asp Gly Arg Thr Ser Tyr Tyr Pro Gly Glu Ala Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val 115 120 215 114 PRT artificial sequence cloned antibody 215 Glu Ser Asp Pro Gly Leu Val Gln Pro Ser Gln Thr Leu Ser Leu Thr 1 5 10 15 Cys Thr Val Ser Gly Gly Ser Leu Arg Ser Asp Asp Tyr Tyr Trp Ser 20 25 30 Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile Ala Tyr Ile 35 40 45 Ser Tyr Thr Gly Gly Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val 50 55 60 Thr Ile Ser Val Asp Thr Ser Arg Asn Gln Phe Ser Leu Arg Leu Arg 65 70 75 80 Ser Val Thr Ala Ala Asp Ser Ala Val Tyr Phe Cys Ala Ser Thr Thr 85 90 95 Ala Val Thr Thr Thr Phe Asp Tyr Trp Gly Arg Gly Thr Leu Val Thr 100 105 110 Val Ser 216 104 PRT artificial sequence cloned antibody 216 Pro Val Gln Pro Leu Glu Phe Thr Phe Thr Asp His Trp Met His Trp 1 5 10 15 Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Leu Ala Arg Ile Asn 20 25 30 Arg Asp Gly Ser Asp Thr Thr Tyr Ala Asp Ser Val Thr Gly Arg Phe 35 40 45 Thr Ile Ser Arg Asp Asn Gly Lys Asn Thr Val Ser Leu Gln Met Asp 50 55 60 Ser Leu Ser Val Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly 65 70 75 80 His His Thr Val Leu Ser Pro Leu Ser Asn Trp Phe Asp Pro Trp Gly 85 90 95 Gln Gly Thr Leu Val Thr Val Ser 100 217 110 PRT artificial sequence cloned antibody 217 Glu Ser Glu Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Thr Trp Val 20 25 30 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr Met Thr Gly 35 40 45 Ser Gly Gly Val Thr Tyr Tyr Ala Asp Val Leu Lys Gly Arg Phe Thr 50 55 60 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 65 70 75 80 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Tyr Gly 85 90 95 Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 218 115 PRT artificial sequence cloned antibody 218 Leu Ala Gly Val Glu Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ala Met His Trp Leu 20 25 30 Arg Gln Ile Pro Gly Lys Gly Leu Gln Trp Val Ser Leu Leu Ser Trp 35 40 45 Asp Gly Val Ser Ala Tyr Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr 50 55 60 Ile Ser Arg Asp Asn Lys Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser 65 70 75 80 Leu Arg Ala Glu Asp Val Ala Leu Tyr Tyr Cys Ala Lys Asp Met Gly 85 90 95 Gly Ala Gln Arg Leu Pro Asp His Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 219 114 PRT artificial sequence cloned antibody 219 Gly Gly Gly Leu Val Gln Pro Gly Ala Ser Val Lys Val Ser Cys Lys 1 5 10 15 Ala Ser Gly Tyr Thr Phe Ser Asp Tyr Phe Met His Cys Val Arg Gln 20 25 30 Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Leu Val Asn Pro Thr Asn 35 40 45 Gly Tyr Thr Ala Tyr Ala Pro Lys Phe Gln Gly Arg Val Thr Met Thr 50 55 60 Arg Gln Arg Phe Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg 65 70 75 80 Ser Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg Val Lys Ser Ser Asp 85 90 95 Ser Ile Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val 100 105 110 Ser Ser 220 103 PRT artificial sequence cloned antibody 220 Arg Cys Pro Ala Lys Leu Leu Asp Thr Pro Phe Ser Val Tyr Phe Met 1 5 10 15 His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Leu 20 25 30 Val Asn Pro Thr Asn Gly Tyr Thr Ala Tyr Ala Pro Lys Phe Gln Gly 35 40 45 Arg Val Thr Met Thr Arg Gln Arg Phe Thr Ser Thr Val Tyr Met Glu 50 55 60 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg 65 70 75 80 Val Lys Ser Ser Asp Ser Ile Asp Ala Phe Asp Ile Trp Gly Gln Gly 85 90 95 Thr Met Val Thr Val Ser Ser 100 221 103 PRT artificial sequence cloned antibody 221 Arg Cys Pro Ala Lys Leu Leu Asp Thr Pro Ser Gly Asp Tyr Phe Met 1 5 10 15 His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Leu 20 25 30 Val Asn Pro Thr Asn Gly Tyr Thr Ala Tyr Ala Pro Lys Phe Gln Gly 35 40 45 Arg Val Thr Met Thr Arg Gln Arg Phe Thr Ser Thr Val Tyr Met Glu 50 55 60 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys Ala Arg 65 70 75 80 Val Lys Ser Ser Asp Ser Ile Asp Ala Phe Asp Ile Trp Gly Gln Gly 85 90 95 Thr Met Val Thr Val Ser Ser 100 222 115 PRT artificial sequence cloned antibody 222 Ser Gly Gly Leu Val Gln Arg Gly Ala Lys Val Leu Arg Leu Ser Cys 1 5 10 15 Val Ala Ser Gly Phe Thr Phe Ser Ser Ser Ala Met Ser Trp Val Arg 20 25 30 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Ser Gly Asn 35 40 45 Gly Phe Ser Thr Tyr Tyr Ala Asp Ser Val Lys Arg Phe Thr Ile Ser 50 55 60 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 65 70 75 80 Ala Glu Asp Thr Ala Glu Tyr Tyr Cys Thr Lys Val Lys Tyr Gly Ser 85 90 95 Gly Ser His Phe Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 223 83 PRT artificial sequence cloned antibody 223 Leu Gly Ser Pro Tyr Ser Ser Ser Ala Met Ser Trp Val Arg Gln Ala 1 5 10 15 Pro Gly Lys Gly Leu Glu Xaa Val Ser Phe Ile Ser Xaa Asn Gly Leu 20 25 30 Ser Ala Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 35 40 45 Asp Asn Ser Xaa Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ser 50 55 60 Glu Asp Thr Ala Glu Tyr Tyr Cys Val Lys Val Xaa Tyr Gly Ser Arg 65 70 75 80 Ser His Phe 224 115 PRT artificial sequence cloned antibody 224 Val Glu Ser Gly Gly Val Val Gln Pro Gly Ala Lys Val Leu Arg Leu 1 5 10 15 Ser Cys Ala Ala Ser Gly Phe Ser Phe Glu Asp Tyr Ala Met His Trp 20 25 30 Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Ser 35 40 45 Trp Asp Val Ile Ser Ala Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 50 55 60 Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr Leu Gln Met Asp 65 70 75 80 Ser Leu Arg Pro Glu Asp Ser Gly Leu Tyr Tyr Cys Gly Arg Asp Ile 85 90 95 Gly Gln Gln Arg Thr Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 225 98 PRT artificial sequence cloned antibody 225 Ala Ala Ser Gly Phe Ile Phe Asp Asp Phe Ala Met His Trp Phe Gln 1 5 10 15 Ala Val Pro Gly Lys Gly Leu Gln Trp Val Gly Leu Met Ser Trp Asp 20 25 30 Gly Val Ser Ala Tyr Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr Ile 35 40 45 Ser Arg Asp Asn Lys Lys Asn Ala Leu Tyr Leu Gln Met Asn Ser Leu 50 55 60 Gly Val Glu Asp Thr Ala Leu Tyr Phe Cys Ala Lys Asp Met Gly Gly 65 70 75 80 Gly Leu Arg Phe Pro His Phe Trp Gly Gln Gly Thr Pro Val Thr Val 85 90 95 Ser Ala 226 111 PRT artificial sequence cloned antibody 226 Phe Trp Leu Gly Gly Pro Trp Arg Leu Ser Cys Ala Val Ser Gly Tyr 1 5 10 15 Thr Leu Ser Ser Ser Ala Met Ile Trp Val Arg Gln Pro Pro Gly Lys 20 25 30 Gly Leu Glu Phe Val Ser Val Ile Ser Gly Asn Gly Leu Ser Ala Tyr 35 40 45 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 50 55 60 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 65 70 75 80 Ala Glu Tyr Tyr Cys Val Lys Val Lys Tyr Gly Ser Arg Ser His Phe 85 90 95 Phe Phe Asp Ser Trp Gly Gln Gly Thr Leu Val Ser Val Ser Pro 100 105 110 227 115 PRT artificial sequence cloned antibody 227 Gly Gly Gly Leu Val Gln Pro Gly Ala Ser Leu Arg Leu Ser Cys Val 1 5 10 15 Ala Ser Gly Phe Thr Leu Ser Ser Ser Ala Met Ser Cys Val Arg Gln 20 25 30 Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ser Ser Gly Asn Gly 35 40 45 Phe Ser Ala Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 50 55 60 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Val 65 70 75 80 Ala Glu Asp Thr Ala Glu Tyr Tyr Cys Thr Lys Val Asn Tyr Gly Ser 85 90 95 Arg Ser His Phe Tyr Phe Gly Ser Trp Gly His Gly Thr Leu Val Ile 100 105 110 Val Ser Ser 115 228 114 PRT artificial sequence cloned antibody 228 Trp Gly Arg Arg Gly Pro Ala Trp Gly Val Pro Val Gly Ser Pro Val 1 5 10 15 Gln Pro Leu Gly Tyr Thr Phe Asp Asp Tyr Ala Met His Trp Leu Arg 20 25 30 Gln Ile Pro Gly Lys Gly Leu Gln Trp Val Ser Leu Leu Ser Trp Asp 35 40 45 Gly Val Ser Ala Tyr Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr Ile 50 55 60 Ser Arg Asp Asn Lys Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu 65 70 75 80 Val Ala Glu Asp Thr Ala Leu Tyr Phe Cys Ala Lys Asp Met Gly Gly 85 90 95 Ala Gln Arg Leu Pro Asp His Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser 229 115 PRT artificial sequence cloned antibody 229 Trp Thr Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Val Ser 1 5 10 15 Val Ala Ala Ser Gly Tyr Thr Phe Asp Asp Tyr Ala Met His Trp Leu 20 25 30 Arg Gln Ile Pro Gly Lys Gly Leu Gln Trp Val Ser Leu Leu Ser Trp 35 40 45 Asp Gly Val Ser Ala Tyr Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr 50 55 60 Ile Ser Arg Asp Asn Xaa Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser 65 70 75 80 Leu Ile Ala Glu Asp Thr Ala Leu Tyr Phe Cys Ala Lys Asp Met Gly 85 90 95 Gly Ala Gln Arg Leu Pro Asp His Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 230 120 PRT artificial sequence cloned antibody 230 Ala Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu 1 5 10 15 Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr Thr Leu Ser Trp 20 25 30 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser 35 40 45 Thr Asp Gly Ser Thr Ile Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe 50 55 60 Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser Leu Gln Met Ile 65 70 75 80 Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val Phe 85 90 95 Phe Gly Gly Asn Phe Arg Ala His Trp Tyr Phe Asp Leu Trp Gly Arg 100 105 110 Gly Thr Leu Val Ala Val Ser Ser 115 120 231 47 DNA artificial sequence primer 231 agaatttgac tagttggcaa gaggcacgtt cttttctttg ttgccgt 47
Claims (9)
1. An engineered nucleic acid strand comprising a predetermined sequence at a first end thereof, a sequence complementary to the predetermined sequence at the other end thereof, and a hairpin structure therebetween.
2. A library of polypeptides produced by a method comprising the steps of:
a) annealing a primer to a template nucleic acid sequence, the primer having a first portion which anneals to the template and a second portion of predetermined sequence;
b) synthesizing a polynucleotide that anneals to and is complementary to the portion of the template adjacent to the location at which the first portion of the primer anneals to the template, the polynucleotide having a first end and a second end, wherein the first end incorporates the primer;
c) separating the polynucleotide synthesized in step (b) from the template;
d) annealing a nested oligonucleotide to the second end of the polynucleotide synthesized in step (b), the nested oligonucleotide having a first portion that anneals to the second end of the polynucleotide, and a second portion having a hairpin structure;
e) extending the polynucleotide synthesized in step (b) to provide an extended polynucleotide comprising a portion that is complementary to the hairpin structure and a terminal portion that is complementary to the predetermined sequence; and
f) amplifying the extended polynucleotide using a single primer having the predetermined sequence.
3) A library as in claim 2 wherein the polypeptides comprise at least a portion of anitbodies.
4) A library of polypeptides produced by a method comprising the steps of:
a) annealing a primer and a boundary oligonucleotide to a template nucleic acid sequence, the primer having a first portion which anneals to the template and a second portion of predetermined sequence;
b) synthesizing a polynucleotide that anneals to and is complementary to the portion of the template between the location at which the first portion of the primer anneals to the template and the portion of the template to which the boundary oligonucleotide anneals, the polynucleotide having a first end and a second end, wherein the first end incorporates the primer;
c) separating the polynucleotide synthesized in step (b) from the template;
d) annealing a nested oligonucleotide to the second end of the polynucleotide synthesized in step (b), the nested oligonucleotide having a first portion that anneals to the second end of the polynucleotide and a second portion having a hairpin structure;
e) extending the polynucleotide synthesized in step (b) to provide an extended polynucleotide comprising a portion that is complementary to the hairpin structure and a terminal portion that is complementary to the predetermined sequence; and
f) amplifying the extended polynucleotide using a single primer having the predetermined sequence.
5) A library as in claim 4 wherein the polypeptides comprise at least a portion of antibodies.
6) A library of polypeptides produced by a method comprising the steps of:
a) annealing an oligo dT primer and a boundary oligonucleotide to an mRNA template;
b) synthesizing a polynucleotide that anneals to and is complementary to the portion of the template between the location at which the first portion of the primer anneals to the template and the portion of the template to which the boundary oligonucleotide anneals, the polynucleotide having a first end and a second end, wherein the first end incorporates the primer;
c) separating the polynucleotide synthesized in step (b) from the template;
d) annealing a nested oligonucleotide to the second end of the polynucleotide synthesized in step (b), the nested oligonucleotide having a first portion that anneals to the second end of the polynucleotide, and a second portion having a hairpin structure;
e) extending the polynucleotide synthesized in step (b) to provide an extended polynucleotide comprising a portion that is complementary to the hairpin structure and a poly A terminal portion; and
f) amplifying the extended polynucleotide using a single primer.
7) A library as in claim 6 wherein the polypeptides comprise at least a portion of antibodies.
8) A library of polypeptides produced by a method comprising:
a) annealing a primer to a family of related nucleic acid sequence templates, the primer having a first portion which anneals to the templates and a second portion of predetermined sequence;
b) synthesizing polynucleotides that anneal to and are complementary to the portion of the templates adjacent to the location at which the first portion of the primer anneals to the templates, the polynucleotides having a first end and a second end, wherein the first end incorporates the primer;
c) Separating the polynucleotides synthesized in step (b) from the templates;
d) Annealing a nested oligonucleotide to the second end of the polynucleotides synthesized in step (b), the nested oligonucleotide having a first portion that anneals to the second end of the polynecleotides, and a second portion having a hairpin structure;
e) Extending the polynucleotides synthesized in step (b) to provide an extended polynucleotide comprising a portion that is complementary to the hairpin structure and a terminal portion that is complementary to the predetermined sequence; and
f) amplifying the extended polynucleotides using a single primer having the predetermined sequence.
9) A library as in claim 8 wherein the polypeptides comprise at least a portion of antibodies.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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US10/628,109 US20040101886A1 (en) | 2000-12-11 | 2003-07-28 | Nested oligonucleotides containing a hairpin for nucleic acid amplification |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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US25466900P | 2000-12-11 | 2000-12-11 | |
US32340001P | 2001-09-19 | 2001-09-19 | |
US10/014,012 US6919189B2 (en) | 2000-12-11 | 2001-12-10 | Nested oligonucleotides containing a hairpin for nucleic acid amplification |
US10/628,109 US20040101886A1 (en) | 2000-12-11 | 2003-07-28 | Nested oligonucleotides containing a hairpin for nucleic acid amplification |
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US10/014,012 Division US6919189B2 (en) | 2000-12-11 | 2001-12-10 | Nested oligonucleotides containing a hairpin for nucleic acid amplification |
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US20040101886A1 true US20040101886A1 (en) | 2004-05-27 |
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US10/014,012 Expired - Lifetime US6919189B2 (en) | 2000-12-11 | 2001-12-10 | Nested oligonucleotides containing a hairpin for nucleic acid amplification |
US10/628,109 Abandoned US20040101886A1 (en) | 2000-12-11 | 2003-07-28 | Nested oligonucleotides containing a hairpin for nucleic acid amplification |
US12/378,811 Expired - Fee Related USRE41365E1 (en) | 2000-12-11 | 2009-02-18 | Nested oligonucleotides containing a hairpin for nucleic acid amplification |
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US10/014,012 Expired - Lifetime US6919189B2 (en) | 2000-12-11 | 2001-12-10 | Nested oligonucleotides containing a hairpin for nucleic acid amplification |
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US12/378,811 Expired - Fee Related USRE41365E1 (en) | 2000-12-11 | 2009-02-18 | Nested oligonucleotides containing a hairpin for nucleic acid amplification |
Country Status (5)
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US (3) | US6919189B2 (en) |
EP (1) | EP1366191A2 (en) |
AU (1) | AU2002230734A1 (en) |
CA (1) | CA2436693A1 (en) |
WO (1) | WO2002048401A2 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050208627A1 (en) * | 2003-09-18 | 2005-09-22 | Bowdish Katherine S | Elicitation of antibodies to self peptides in mice by immunization with dendritic cells |
US20070054378A1 (en) * | 2003-09-18 | 2007-03-08 | Alexion Pharmaceuticals, Inc. | Elicitation of antibodies to self peptides in mice by immunization with dendritic cells |
US9890414B2 (en) | 2012-11-28 | 2018-02-13 | Abwiz Bio, Inc | Preparation of gene-specific templates for the use in single primer amplification |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2450217A1 (en) * | 2001-05-14 | 2002-11-21 | Henry Hongjun Ji | Novel method for cloning variable domain sequences of immunological gene repertoire |
WO2003025202A2 (en) * | 2001-09-19 | 2003-03-27 | Alexion Pharmaceuticals, Inc. | Engineered templates and their use in single primer amplification |
ATE466107T1 (en) | 2004-07-01 | 2010-05-15 | Gen Probe Inc | METHODS AND COMPOSITIONS FOR DETECTING NUCLEIC ACIDS IN A BIOLOGICAL SAMPLE |
US7833716B2 (en) | 2006-06-06 | 2010-11-16 | Gen-Probe Incorporated | Tagged oligonucleotides and their use in nucleic acid amplification methods |
WO2010068473A1 (en) | 2008-11-25 | 2010-06-17 | Gen-Probe Incorporated | Compositions and methods for detecting small rnas, and uses thereof |
CN106811459B (en) * | 2015-11-27 | 2019-09-27 | 清华大学 | A method of it is checked using short strand primer and improves DNA clone and packaging efficiency |
Family Cites Families (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
BR8108820A (en) | 1980-09-24 | 1982-08-24 | Cetus Corp | DIAGNOSTIC PROCESS AND PROBE |
US4582788A (en) | 1982-01-22 | 1986-04-15 | Cetus Corporation | HLA typing method and cDNA probes used therein |
EP0084796B1 (en) | 1982-01-22 | 1990-05-02 | Cetus Corporation | Hla typing method and cdna probes used therein |
US4683194A (en) | 1984-05-29 | 1987-07-28 | Cetus Corporation | Method for detection of polymorphic restriction sites and nucleic acid sequences |
US4683202A (en) | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
CA1284931C (en) | 1986-03-13 | 1991-06-18 | Henry A. Erlich | Process for detecting specific nucleotide variations and genetic polymorphisms present in nucleic acids |
CA1338457C (en) | 1986-08-22 | 1996-07-16 | Henry A. Erlich | Purified thermostable enzyme |
US5130238A (en) | 1988-06-24 | 1992-07-14 | Cangene Corporation | Enhanced nucleic acid amplification process |
JP2919890B2 (en) | 1988-11-11 | 1999-07-19 | メディカル リサーチ カウンスル | Single domain ligand, receptor consisting of the ligand, method for producing the same, and use of the ligand and the receptor |
CA2016842A1 (en) | 1989-05-16 | 1990-11-16 | Richard A. Lerner | Method for tapping the immunological repertoire |
US5595891A (en) | 1990-07-19 | 1997-01-21 | Behringwerke Ag | Method for producing a polynucleotide for use in single primer amplification |
DE69310179T2 (en) * | 1992-07-31 | 1997-07-31 | Behringwerke Ag | METHOD FOR INTRODUCING DEFINED SEQUENCES AT THE 3 'END OF POLYNUCLEOTIDES |
FR2724934B1 (en) | 1994-09-26 | 1997-01-24 | Bio Merieux | CHIMERIC OLIGONUCLEOTIDE AND ITS USE IN OBTAINING NUCLEIC ACID TRANSCRIPTS |
WO1997004131A1 (en) * | 1995-07-21 | 1997-02-06 | Forsyth Dental Infirmary For Children | Single primer amplification of polynucleotide hairpins |
US5962271A (en) | 1996-01-03 | 1999-10-05 | Cloutech Laboratories, Inc. | Methods and compositions for generating full-length cDNA having arbitrary nucleotide sequence at the 3'-end |
US6087099A (en) * | 1997-09-08 | 2000-07-11 | Myriad Genetics, Inc. | Method for sequencing both strands of a double stranded DNA in a single sequencing reaction |
US6277607B1 (en) * | 1999-05-24 | 2001-08-21 | Sanjay Tyagi | High specificity primers, amplification methods and kits |
US6251639B1 (en) | 1999-09-13 | 2001-06-26 | Nugen Technologies, Inc. | Methods and compositions for linear isothermal amplification of polynucleotide sequences, using a RNA-DNA composite primer |
US6582938B1 (en) * | 2001-05-11 | 2003-06-24 | Affymetrix, Inc. | Amplification of nucleic acids |
WO2003025202A2 (en) * | 2001-09-19 | 2003-03-27 | Alexion Pharmaceuticals, Inc. | Engineered templates and their use in single primer amplification |
-
2001
- 2001-12-10 EP EP01990978A patent/EP1366191A2/en not_active Withdrawn
- 2001-12-10 WO PCT/US2001/047727 patent/WO2002048401A2/en not_active Application Discontinuation
- 2001-12-10 US US10/014,012 patent/US6919189B2/en not_active Expired - Lifetime
- 2001-12-10 AU AU2002230734A patent/AU2002230734A1/en not_active Abandoned
- 2001-12-10 CA CA002436693A patent/CA2436693A1/en not_active Abandoned
-
2003
- 2003-07-28 US US10/628,109 patent/US20040101886A1/en not_active Abandoned
-
2009
- 2009-02-18 US US12/378,811 patent/USRE41365E1/en not_active Expired - Fee Related
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050208627A1 (en) * | 2003-09-18 | 2005-09-22 | Bowdish Katherine S | Elicitation of antibodies to self peptides in mice by immunization with dendritic cells |
US20070054378A1 (en) * | 2003-09-18 | 2007-03-08 | Alexion Pharmaceuticals, Inc. | Elicitation of antibodies to self peptides in mice by immunization with dendritic cells |
US9890414B2 (en) | 2012-11-28 | 2018-02-13 | Abwiz Bio, Inc | Preparation of gene-specific templates for the use in single primer amplification |
Also Published As
Publication number | Publication date |
---|---|
USRE41365E1 (en) | 2010-06-01 |
US20050079489A1 (en) | 2005-04-14 |
WO2002048401A3 (en) | 2003-09-12 |
WO2002048401A2 (en) | 2002-06-20 |
US6919189B2 (en) | 2005-07-19 |
EP1366191A2 (en) | 2003-12-03 |
AU2002230734A1 (en) | 2002-06-24 |
CA2436693A1 (en) | 2002-06-20 |
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AS | Assignment |
Owner name: ALEXION PHARMACEUTICALS, INC., CONNECTICUT Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BOWDISH, KATHERINE S.;FREDERICKSON, SHANA;MCWHIRTER, JOHN;AND OTHERS;REEL/FRAME:017611/0672;SIGNING DATES FROM 20020707 TO 20020711 |
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STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |