CA1171371A - Oligonucleotides useful as adaptors and adapted molecules for cloning dna and method of employing same - Google Patents
Oligonucleotides useful as adaptors and adapted molecules for cloning dna and method of employing sameInfo
- Publication number
- CA1171371A CA1171371A CA000313650A CA313650A CA1171371A CA 1171371 A CA1171371 A CA 1171371A CA 000313650 A CA000313650 A CA 000313650A CA 313650 A CA313650 A CA 313650A CA 1171371 A CA1171371 A CA 1171371A
- Authority
- CA
- Canada
- Prior art keywords
- cloning vehicle
- dna
- molecule
- adaptor
- recognition site
- 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.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
Abstract
Abstract of the Disclosure Synthetic oligonucleotides have been designed and prepared which are useful in the molecular cloning of a variety of DNA molecules. By means of such oligonucleotides, genetic informational material, e.g., DNA, can be joined to a cloning vehicle and transferred into host cells by trans-formation. Additionally, a method for determining whether genetic informational material has been transferred into transformation host cells has been developed.
Description
:~17137 1 !
Background of the Invention Molecular cloning has become a powerful tool for the ampliEication of specific D~A (deoxyribonucleic acid) fragments and their subsequent isolation in high yields.
Two basic steps are involved in molecular cloning. First the DNA fragments to be cloned are joined in vitro to an autonomously replicating cloning vehicle molecule, e.g., plasmid DNA [Cohen, S.N. et al., PNAS 70, 3240 ~1973);
Tanaka, T. and Weisblum, B., J. Bacterioloqy 121, 354 (1975)] or ~ phage DN~ [Thomas, M. et al., PNAS 71, 4579 ~1974); rlurray, N.E. and ~lurray, K., Nature 251, 476 (1974)].
The hybrid recombinant DNA-cloning vehicles so formed are then introduced into host cells, e.g., E. coli cells, by transformation and cloned by a suitable technique such as single colony isolation or plaque formatio~.
In one cloning method, two different DNA molecules are cut by the same restriction endonuclease to produce identical cohesive ends. The DNA molecules are annealed to one another and then covalently joined by DNA ligase. This method limits the size and kind of DN~ fragments that can be cloned since it often requires cloning of a much larger DNA
fragment than one is interested in. For example, if one wants to clone a small DNA fra~ment such as a promoter (e.g., an RNA
polymerase protected fragment), the nearest restriction endo-nuclease sites may be relatively distant, and thus extraneous D~IA sequences must be included in the cloned DNA. This creates the possibility that undesirable or even hazardous sequences may be transferred, and it is this possibility which has led to public interest in the entire area of molecular cloning and recombinant DNA research~ Furthermore~ many DN~ fragments cannot be cloned by this method because of the lack o a suitable restriction enzyme for producing molecules with appropriate cohesive ends.
The present invention utilizes chemically synthesized oligonucleotides having nucleotide sequences which are the recognition sites for restriction erdonucleases as adaptor molecules. These adaptor molecules are joined at ~:
the ends of natural or synthetic DNA molecules to form adapted DNA molecules. The ends of such natural or synthetic DNA molecules can be even-ended or can have a protruding nucleotide sequence. Al~ernatively, adapted DN~ molecules which comprise synthetic DNA molecules having such adapto~
molecules incorporated therein at their ends are prepared.
Such adapted DNA molecules are then joined to a cloning vehicle~
thereby making the cloning procedure much more selective, versatile and safe. A part of the substance of this invention has been described recently in two publications ~Bahl, Chander P., et al., Gene 1, 81 (1976? and Marians, K.J. et al., Nature, 263, 744 (197633.
"
Brief Summary of the Invention .
This invention concerns a novel approach to the molecular cloning of a variety of DNA molecules. In one aspect of the invention, adaptor molecules useful for the insertion of genetic informatlonal material, e.g., DN~, into a cloning vehicle have been defined and methods of preparing such adaptor molecules have been developed. In another aspect ~ r~
1 1~137~
, of the invention, adapted DNA molecules have been defined which comprise genetic informational material, e.g., DNA, for insertion into a cloning vehicle and adaptor molecules joined at both ends to the genetic informational material being inserted. Methods of preparing such adapted DNA
molecules have also been developed. In still another aspect of the invention, modified cloning vehicles which contain such adapted DNA molecules have been defined and a method of preparing such modified cloning vehicles has heen developed. In yet another aspect of the invention, such modi~ied cloning vehicles are used to transform host cells and thus transfer genetic informational material such as DNA into host cells. Finally, the invention describes a method for determining whether genetic informational material has been transferred into cells by using an indicator DNA
such as the lac operator as part of the genetic informational material in an adapted DNA molecule, forming a modified cloning vehicle which includes such an adapted DNA molecule, transforming the host cells using the modified cloning vehicle and screening the transformed cells to determine whether transfer has taken place.
These and other aspects of the invention are set forth more fully in the detailed decription of the invention and the claims which follow.
:~713~1 Detailed Description of the Invention Adaptor Molecules Adaptor molecules for the insertion of genetic informational material, e.g., DNA, into cloning vehicles have been prepared. These adaptor molecules are either single-stranded or double-stranded oligonucleo-tides. If double-stranded, adaptor molecules may have either one pro-truding nucleotide ~equence which is a recognition site for a restriction endonuclease at one end of the duplex or two protruding nucleotide sequences which are recognition sites for the same or different restriction endonucleases at opposite ends of the duplex.
Examples of protruding nucleotide sequences which are reco~nition sites for restriction enzymes include: 5' pA-A-T-T, the partial recognition site or EcoRI res-triction endonuclease; 5' pG-A-T-C, the partial recognition site for B mI res~riction endonuclease; and 5' pA-G-C-T, the paxtial recognitio~ site for HindIII restriction~endonuclease.
Similar adaptor molecules with partial recognition sites fox other restriction endonucleases such as PstI, SalI, HaeII, XmaI and ~II can also be synthesiæed and used or cloning.
The adaptor molecules of the inventio are prepared in a number of ways. First, duplex adaptor molecules having one protruding nucleotide sequence may be prepared by chemically synthesizing single-stranded oligonucleotides which include th~
recognition site for a restriction endonuclease and which are self-complementary. A double-stranded duplex is then prepared from the self-complementary oligonucleotides and the duplex is ~ ~1371 then contacted with the restriction endonuclease whose recognition site is included in the oligonucleotides.
The restriction endonuclease digests the duplex in such a way that an adaptor molecule having a protruding nucleo-tide seyuence which is the recognition site for the restriction endonuclease results.
For example, two self-complementary decadeoxyribo-nucleotides d(C-C-G-G-A-T-C-C-G-G) (BamI adaptor sequence including the BamI restriction endonuclease recognition site, 5' pG G-A-T-C-C) and d(A-C-A-A-G-C-T-T-G-T) (HindIII adaptor sequence including the IIindIII restriction endonuclease recognition site, 5' pA-A-G-C-T-T) were synthesized by the improved phosphotriester method developed previously ~Itakura, K. et al., Canadian J. Chem. 51, 3649 (1973~;
Itakura, K. et al., J. Am. Chem. Soc. 97, 7327 (1975);
Katagiri, N. et al., J. Am. Chem. Soc. 97, 7332 (1975);
Bahl, C.P., et al., Gene 1, 81 (1976); Stawinsky, J., et al., A Nucleic Acids Res. 4, ~ (1977)]~ Two new steps were intro-duced in these syntheses: the dimethoxytrityl group was removed by a 2% solution of benzenesulfonic acid in chloro-form and the chlorophenyl phosphate protecting group was removed by treatment with concentrated ammonium hydroxide for 4-6 hours. The oligonucleotides were characterized by two-dimensional electrophoresis-homochromatography of their partial venom phosphodiesterase digestion products ~Jay, E., et al., Nucleic Acids Res. 1, 331 (1974) and Tu, C.D., et al., A _ . Biochem 74, 7~ (1976)] which verified the above sequences of the t~lo synthetic decadeoxynucleotides.
~1~1137~
Double-stranded duple~es were prepared from these two self-complèmentary ol.igonucleotides. Specifically, the chemically synthesized decadeoxynucleotides (~00 pmoles) were phosphorylated at the 5' end using ~y32p] ATP and polynucleo-tide kinase [Wu, R. et al., Methods in Cancer Res., 12, 88 (1976)]. The labeled decadeoxynucleotides were dissolved in a suitable amount (about 100~1) of a suitable buffer such as 100 n~l Tris-HCl (pH 7.5) heated to about 90C. for about 1 minute, quickly chilled to about 0C. and then incubated at about 70C. for about 30 minutes. The duplexes of the deca-deoxynucleotides were forriled by slowly cooling the samples to about roorn temperature and then to about 4C.
The duplex which i.ncluded the recognition site for BamI restriction endonuclease was then contacted with this enzyme [Wilson, &.A. and Young, F.E., J. Mol. Biol. 97, 123 ~1975)] and after dlgestion gave an adaptor molecule having the protruding sequence 5' pG-A-T-C. Thus, 5' pC-C-G~G~A-T-C--C-G-G
3' G-G-C-C~-T-A-G-G-C-Cp ~ BamI endonuclease 5' C-C-G
3' G-G-C-C-T-A-Gp . A second method by which duplex adaptor molecules having one protruding nucleotide sequence may be prepared involves the chemical synthesis of a first single-stranded oli.gonucleotide which includes the recognition site for a restriction endonuclease and a second single-stranded oligonucleotide which does not include the recognition site for the restriction endonucléase but is otherwise complementary to the first, that is, one oligonucleotide is longer than the ~17~ 371 other and the nucleotides which correspond to the recognition site of the restriction endonuclease are protruding. Upon formin~ a double-stranded dupleY~ from the oligonucleotides, an adaptor molecule results in which the recognition site protrudes.
For example, an oligonucleotide having the sequence 5' pG-A-~-C-C-C-C-G-G-G can be synthesized by the method described previously, and similarly an oligonucleotide having the sequence 5' pC-C-C-G-G-G can be synthesized. A
duplex of these oligonucleotides can be formed as previously described which results in an adaptor molecule having the protruding sequence for BamI restriction endonuclease, namely, 5' pG-A-T-C. This type of adaptor can be defined as a ready-made adaptor. The following is illustrativeD
BamI site 5' pG-A-T-C-C-C-C-G-G-G
3' ~-G-5-C-C-C
Duplex adaptor molecules having two protruding nucleotide sequences which are recognition sites for the same or different restriction endonucleases can be prepared in a similar way. Single-stranded oligonucleotides which include the recognition site for a restriction endonuclease and are partially complementary are chemically synthesi~ed.
A double-stranded duplex is then formed from the oligonucleo-tides in which the nucleotide sequences which are recognition sites for the same or diffe~ent restriction endonucleases protrude at opposite ends of the duplex.
~ 17~ 3~
Conversion Adaptor Molecules Duplex conversion adaptor molecules having t~o pro-truding nucleotide sequences which are recognition sites for t~o different restriction endonucleases can be prepared by chemical synthesis of two single-stranded oligonucleotides which are partially complementary. A partially double-stranded duple~ is then formed from the ollgonucleotides in wllich the nucleotide sequences which are recognition sites for the two different restriction endonucleases protrude at opposite ends of the duplex~ The following example is illustrative:
BamI site 5' pG-A-T-C-C-C-C-G-G-G 3' G-G-G-C-C-C-T~T-A-Ap 5' ,t EcoRI site A conversion adaptor molecule with two different protruding nucleotide sequences can also be made by joining two dif~erent even-ended adaptor molecules using polynucleo-tide ligase, ~ollowed b~ digestion with the two appropriate restriction endonucleases~
A second type of conversion adaptor molecule, a completely single-stranded oligodeoxynucleotide, having two recognition sites for two different restriction endonucleases can be prepared in a similar way. This type of conversion adaptor molecule is used to convert a 3' protruding nucleo-tide sequence at the termini of a DNA molecule to be cloned to a 5' protruding sequence, or vice versa. For example, ~aeII restriction endonuclease digests DNA to give 3' protrud-ing ends, 3' d~C-G-C-G). In order to clone a DNA molecule such as DNA-X ~ith 3' protruding d(C-G-C-G) ends, the conversion adaptor can be joined to each end of DNA-X to produce a DNA
3 7 ~
molecule containing protruding 5' A-A-T-T ends. The latter can then be joined to the same 5' A-A-T-T ends of the cloning vehicle as follows:
DNA-X EcoRI site-HaeII conversion adaptor 5' G-C-G-C 3' 3' C-G-C-G S' pA-A-T-T-G-C-G-C
HaeII site EcoRI HaeII
- site site ~ T4 ligase 5' pA-A-T-T-G-C-G-C - -- G-C-G-C
3' 1 ~ C-G-C-G - C-G-C-G-T-T-A-Ap EcoRI site ~ ~
- EcoRI site cloning vehicle containing S' protruding pA-A-T-T sequence ~ ~ T4 ligase modified cloning vehicle .
In the event the DNA-X, after cloning, needs to be excised at the EcoRI or HaeII site, then the following conver-sion adaptor can he used:
5' pA-A-T-T-C-A-G-C-G-C
EcoRI site HaeII site Other examples of such conversion adaptors include: EcoRI
site - PstI site, BamI site - HaeII site, ~amI site - PstI
site, HindIII site - HaeII site, and HindIII site - Pst site.
~ daptor molecules of 3 different lengths can be prepared and used to adjust Ihe genetic informational material, e.g., DNA, ~hich is to be cloned to the proper reading frame for protein synthesis. Once the adaptor molecule is joined 1~7137~
to the genetic informational materia]. to be inserted and then inserted into a cloning vehicle and used to transform cells, the expression of the yenetic informational material in terms of RNA and protein synthesis depends upon whether the reading frame is correct. In cases where RNA synthesis is initiated rom a promoter site on the cloning vehicle, the number of nucleotides between the start of the mRNA and the beginning of the coding sequence of ,the DNA molecule should be a multiple of 3 in order to keep the reading frame for protein s~nthe.sis in phase during the translation of the mRNA sequence into protein sequence. If the exact length of genetic informational DNA is unknown, in order to make the length of genetic in~'orma.tional DNA plus the adaptor as multiples of 3, three different lengths of the adaptor (,3n, 3n+1 and 3n+2) must be available. Thus, all of the adaptors discussed must be constructed to give three different lengths. For example, if the adaptor 5' pG-A-T-C-C-C-C-G-G-G 3' G-G-G-C-C-C
is joined to genetic informational DNA to be cloned, it gives six extra nucleotides (multiple of 3, or 3n+0). The adaptor molecule can be converted to give 3n+1 and 3n+2 as follo~s:
5' pG-A-T-C-C-C-C-G-G-G 3' 5' pA-A-A-A 3' G-G-G-C-C-C + T-T-T-T
~ T4 ligase 5' pG-A-T-C-C-C-C-G-G-G-A-A-A-A 3' G-G-G-C-C-C-T-T-T-T
length 3n+1 I1~1371 If (pA)5:(pT)5 is used instead of (pA)~:(pT)4, then after joining it to ~he adaptor the duplex length will be 3n-~2.
In all the adaptors designed so ar, the DNA
sequence which corresponds to the termination codon for protein synthesis (U~, UAG, UGA) is avoided.
Adapted DNA Molecules Adapted DNA molecules can be prepared which include genetic informational material, e.g., DNA, typically in the form of a double-stranded duplex, which is to be inserted into a cloning vehicle and adaptor molecules joined to the ends of the genetic informational material. The genetic informational material can, within the limits imposed by the genetic code, be translated into any polypeptide. It can be naturally derived or synthetically produced.
The adaptor molecules described previously can be joined to opposite ends of genetic informational material which is to be inserted into a cloning vehicle using a poly-nucleotide ligase, e.g., T~ polynocleotide ligase or E. coli polynucleotide ligase. ~he adaptor molecules can be identical or different and can have one or two protruding nucleotide sequences depending upon the nature and structure of the genetic informational material such as DNA to which they are joined. The adaptor molecules are extremely useful tools in molecular clonlng since the same oligonucleotide adaptor molecules can serve to introduce any double-stranded DNA
molecule into cloning vehicles at speci-fic sites. The double-stranded DNA may be obtained by cleavage with a number o~
~12-1 ~137~
restriction endonucleases such as HaeIII and AluI to give even-ended DNA, or the ~uplex DNA may be chemically synthesized.
Alternatively, adapted DNA molecules can be pre-pared by chemically synthesizing a first single-stranded oligonucleotide which includes the genetic informational material to be inserted into a cloning vehicle and a recog-nition site for a restriction endonuc]ease and a second single-stranded oligonucleotide which is partially comple-mentary to the first oligonucleotide and includes a recognition site for a restriction endonuclease, either the same or a different endonuclease. A double-stranded duplex is then formed from the oligonucleotides in which the recognition sites protrude.
Still another method exists for preparing adapted DNA molecules. This involves chemically synthesizing self-complementary single-stranded oligonucIeotides which include the recognition site for a restriction endonuclease. Double-stranded duple~es are then formed from pairs of self-complementary oligonucleotides. The duplexes which are formed can be either identical or dif erent. They are enzymatically joined to opposite ends of the genetic informational matexial to be inserted into a cloning vehicle using a polynucleotide ligase. The resulting molecule is then digested with one or two restriction endonucleases depending upon whether the xecognition sites included in the duplexes were for identisal or different restric-tion endonucleases.
To illustrate this method of preparing an adapted D~A
molecule, the 21 nucleotide-long 12C operator dup]ex ~as .
3. ~137~
chemically synthesized. Two oligonucleotide duplexes were separately synthesized which contained the recognition site ~or a restriction endonuclease, in this case the site for BamI endonuclease. The latter duplexes were then joined to opposite ends of the lac operator using a polynucleotide ligase such as T4 ligase. The resulting molecule ~as then digested with BamI endonuclease which created protruding recognition sequences for BamI endonuclease, namely, 5' pG-A~T-C. The following depicts the reaction sequence:
5' pA-A-T-T-G-T-G-A-G-C-G-G-A-T-A-A-C-A-A-T-T
3' T-T-A-A-C-A-C-T-C-G-C-C-T-A-T-T-G T-T-A-Ap (lac operator) 5' pC-C-G-G-A-T-C-C-G-G
~2 3' G-G-C-C-T-A-G-G-C-Cp (decanucleotide duplex containing site ~ for BamI restriction endonuclease~
5' pC-C-G-G-A-T-C-C-G-G - C-C-G-G-A-m-C-C-G-G
(lac operator~
3' G-G-C-C-T-A-G-G-C-C - G-G-C-C-T-A-G-G-C-Cp BamI endonuclease 5' ~G-A-T-C-C-G-G - C-C-G
3' G-C-C -- G-G-C-C-T-A-Gp (adapted DNA molecule containing lac operator and protruding recognition sites for BamI endonuclease) Alternatively, a lac operator DNA has also been synthesized which included the protruding recognition sites ~or EcoRI éndonuclease [Marians, K.J. et al., Nature ~3, 744 (1976)]. This lac operator DNA can be joined directly to the DNA to be cloned at the EcoRI site, and then to the cloning vehicle.
~17137~
To m~re specifically illustrate, an adap-ted DNA
molecule which included lac operator DNA and protruding nucleotide sequences at both ends which were recot~nition sites for BamI endonuclease was prepared as follows: First, the synthetic decadeoxyribonucleotide duplex which included the recognition site for _ mI endonuclease was prepared as described previously. The 21 nucleotide-long synthetic lac operator was prepared. [Bahl, C.P. et al., PNAS 73, 91 (1976~] ~hen the synthetic duplex (about 10 pmole) and the synthetic lac operator (about 1.0 pmole) were joined end-to-end [Sgaramella, V. et al., PNAS 67, 1468 (1970)~ by incubating with about 3 units of T~ DNA ligase in about 50 ~1 of a solu-tion containing about 20~M Tris-~lCl (pH 7.5), about lOmM dithio-threitol, about lOmM MgC12 and about 35 ~M ~TP at about 20C.
for about 6 hours. The solution was heated to about 70C. for about 5 minutes to inactivate the ligase and cooled slowly to room temperature, 2 volumes of ethanol were added, and after about 12 hours at about 20~C the DNA was pelleted at about 10,000 ~ or about 1 hour. The pellet was dissolved in about 50 ~1 of a solution containing about 6.6mM Tri~-HCl (pH 7.5~ t about 6.6mM ~gC12 and about lmM dithiothreitol~ To this solu-tion was added 2 units of BamI endonuclease. The sample was incubated at about 37C. for about 12 hours to produce the adapted DNA molecule.
Modified Cloning Vehicles _ _ Modified cloning vehicles can be prepared from a cloning vehicle such as plasmid, phage or viral DNA which has been contacted with a restriction endonuclease so that a pro-truding oligonucleotide complementary to the recognition site -1 1 7 :1 3 ~
for the restriction enzyme has resulted by joining to such an endonuclease-treated cloning vehicle an adapted DNA
molecule such as has been described hereinabove. Specific cloning vehicles include p~lB9 plasmid DNA and ~ phage DNA.
The adapted DNA molecule and the endonuclease-treated cloning vehicle can be enzyma~ically joined by use of a polynucleotide ligase such as T4 polynucleotide ligase or E. coli. polynucleo-tide ligase. If the restriction endonuclease site at the termini of the cloning vehicle is different from that of the DNA molecule to be cloned, either the cloning vehicle or the DNA to be cloned can be modified by the use of adaptors.
By this method, synthetic lac operator DNA has been inserted at the BamI, HindIII and EcoRI sites o pMB9 DNA.
For example, linear pMB9 DNA prepared by contacting pMB9 DNA
with BamI restriction endonuclease and an adapted DNA molecule which included lac operator DNA and protruding nucleotide sequences at both ends which were recognition sites or BamI
endonuclease were heated to about 70C. for about 5 minutes~
cooled slowly to room temperature, and the DNA was precipitated by adding 2 volumes of ethanol and after about 12 hours at about 20C. the DNA was pelleted at about 10,000 g for about 1 hour.
The DNA pellet was dissolved in about 50 ~1 of a solution contain-ing about 20mM Tris-HC1 (pH 7.5), about lOmM MgC12, about lOmM
dithiothreitol and about 35 ~M ATP. Three units of DNA ligase ere added and the samples were incubated at about 12.5C. for ahout 24 hours to produce lac-p~9 DNA. After heating at about 70C. for about 5 minutes, and slowly cooling to roo~ tempera-ture, the lac-pMB9 DNA was used directly for transformation as described below.
~17~ ~7~
This method is also suitable for the insertion of mul-tiple copies of a gene such as lac opera~or DNA into a cloning vehicle or for the insertion of a combination of genes.
In order to characterize the lac-pMB9 DNA, it was isolated after transformation and amplification by the addi-tion of chloramphenicol to the bacterial culture, labelled by nick translation [Maniatis, T. et al., _NAS 72~ 1184 (1975)]
and then studied for lac repressor binding properties. The inhibitory effect of isopropyl thioglactoside (IPTG) showed that the binding was specific, thus confirming that a lac operator had been inserted into the plasmid.
The modified plasmid DNA was further characterized by digestion wi~h the appropriate restriction endonuclease and then labelled at the 3'-ends by repair synthesis in the presence of [~-3 P~ dNTP [Wu, ~. et al., Methods in Cancer ~esearch 12, 88 (1976)]. This gave two fragments on poly-acrylamide gel electrophoresis which corresponded to the linear pMB9 DNA and to the lac operator.
Transformation The transfer of genetic informational material such as DNA into host cells, e.g., E. coli cells, can be effected by transformation using the modified cloning vehicles. For example, the lac p~lB9 DNA was used to transform competent E. coli HB 129 cells as follows: The D~A and r~cipient cells -~ere mixed together and incubated at about 0C. for about 30 minutes. The temperature of the mixture was raised to about ~2C. for about 2 minutes and thenchilled to facilitate upta~e ~ ~137~
of the DNA by the cells. ~bout nine volumes of pre~larmed L-broth were added and the cells allowed to recover at about 37C. for about 2 hours. One volume of L-broth supplemented with about 10 ~g ml l of tetracycline was then added. After an additional approximately 30 minutes at 37C., the tetracycline concentration was brought up to a final level of about 20 ~g ml 1 This cell suspension was used to inoculate 100 ml of M9 medium for the isolation of a larger amount of plasmid DNA [Katz, L. and Helinski, D . R., J . Bact.
119, 450 (1973)]. This plasmid DNA (about 30 ~g) which con-tained some modified lac-pMB9 D~A was enriched for lac sequences by binding it to the lac repressor (about 4.5 ~g) on ~ ~illipore filter and eluting it with about l ml of lmM
~ h/~g~/ac7~osld~
isopropyl thioglactocido (IPTG). This DNA (about 6% of the input), enriched for lac sequences, was used for a subsequent transformation on nutrient agar plates containing about 20 ~g ml of tetracycline~ The frequency of transformation was 6.4 x 10 transformants per ~g of recombinant DNA per viable cell, whereas in the same conditions native pMB9 gave a fre-quency of 1.8 x 10 2 Since about l x 107 viable cells were used, the number of transEormants per ~g of recombinant DNA
was about 6,000.
~lethod for Determining Whether Genetic Informational Material Such as DNA Has Been Transferred In order to determine whether ~enetic informational DNA has been transferred into host cells, an indicator DNA
molecule such as the lac operator can be included as part of the yenetic informational DNA in an adapted D~A molecule.
This cdapted DNA molecule is then inserted into a cloning vehicle to prepare a modified cloning vehicle and host cells are transformed using the modified cloning vehicle. If the ~7137~
lae operator is used, transformed cells are sereened to determine whether transfer has taken plaee by platlng the transformed eells in agar plates containing x-gal which will result in the produetion of blue colonies i~ the genetie informational DNA has been transferred because transformed eells which include the lac operator will be constitutive ~or ~-galactosidase synthesis [Miller, J.H., Experiments in Molecular Geneties, Cold Spring ~arbor Lab. 48 (1972)].
. . _ _ .
As will be obvious to one skilled in the art, many modifications in the invention are possible ~ithout departing from the spirit and seope thereof.
~19-
Background of the Invention Molecular cloning has become a powerful tool for the ampliEication of specific D~A (deoxyribonucleic acid) fragments and their subsequent isolation in high yields.
Two basic steps are involved in molecular cloning. First the DNA fragments to be cloned are joined in vitro to an autonomously replicating cloning vehicle molecule, e.g., plasmid DNA [Cohen, S.N. et al., PNAS 70, 3240 ~1973);
Tanaka, T. and Weisblum, B., J. Bacterioloqy 121, 354 (1975)] or ~ phage DN~ [Thomas, M. et al., PNAS 71, 4579 ~1974); rlurray, N.E. and ~lurray, K., Nature 251, 476 (1974)].
The hybrid recombinant DNA-cloning vehicles so formed are then introduced into host cells, e.g., E. coli cells, by transformation and cloned by a suitable technique such as single colony isolation or plaque formatio~.
In one cloning method, two different DNA molecules are cut by the same restriction endonuclease to produce identical cohesive ends. The DNA molecules are annealed to one another and then covalently joined by DNA ligase. This method limits the size and kind of DN~ fragments that can be cloned since it often requires cloning of a much larger DNA
fragment than one is interested in. For example, if one wants to clone a small DNA fra~ment such as a promoter (e.g., an RNA
polymerase protected fragment), the nearest restriction endo-nuclease sites may be relatively distant, and thus extraneous D~IA sequences must be included in the cloned DNA. This creates the possibility that undesirable or even hazardous sequences may be transferred, and it is this possibility which has led to public interest in the entire area of molecular cloning and recombinant DNA research~ Furthermore~ many DN~ fragments cannot be cloned by this method because of the lack o a suitable restriction enzyme for producing molecules with appropriate cohesive ends.
The present invention utilizes chemically synthesized oligonucleotides having nucleotide sequences which are the recognition sites for restriction erdonucleases as adaptor molecules. These adaptor molecules are joined at ~:
the ends of natural or synthetic DNA molecules to form adapted DNA molecules. The ends of such natural or synthetic DNA molecules can be even-ended or can have a protruding nucleotide sequence. Al~ernatively, adapted DN~ molecules which comprise synthetic DNA molecules having such adapto~
molecules incorporated therein at their ends are prepared.
Such adapted DNA molecules are then joined to a cloning vehicle~
thereby making the cloning procedure much more selective, versatile and safe. A part of the substance of this invention has been described recently in two publications ~Bahl, Chander P., et al., Gene 1, 81 (1976? and Marians, K.J. et al., Nature, 263, 744 (197633.
"
Brief Summary of the Invention .
This invention concerns a novel approach to the molecular cloning of a variety of DNA molecules. In one aspect of the invention, adaptor molecules useful for the insertion of genetic informatlonal material, e.g., DN~, into a cloning vehicle have been defined and methods of preparing such adaptor molecules have been developed. In another aspect ~ r~
1 1~137~
, of the invention, adapted DNA molecules have been defined which comprise genetic informational material, e.g., DNA, for insertion into a cloning vehicle and adaptor molecules joined at both ends to the genetic informational material being inserted. Methods of preparing such adapted DNA
molecules have also been developed. In still another aspect of the invention, modified cloning vehicles which contain such adapted DNA molecules have been defined and a method of preparing such modified cloning vehicles has heen developed. In yet another aspect of the invention, such modi~ied cloning vehicles are used to transform host cells and thus transfer genetic informational material such as DNA into host cells. Finally, the invention describes a method for determining whether genetic informational material has been transferred into cells by using an indicator DNA
such as the lac operator as part of the genetic informational material in an adapted DNA molecule, forming a modified cloning vehicle which includes such an adapted DNA molecule, transforming the host cells using the modified cloning vehicle and screening the transformed cells to determine whether transfer has taken place.
These and other aspects of the invention are set forth more fully in the detailed decription of the invention and the claims which follow.
:~713~1 Detailed Description of the Invention Adaptor Molecules Adaptor molecules for the insertion of genetic informational material, e.g., DNA, into cloning vehicles have been prepared. These adaptor molecules are either single-stranded or double-stranded oligonucleo-tides. If double-stranded, adaptor molecules may have either one pro-truding nucleotide ~equence which is a recognition site for a restriction endonuclease at one end of the duplex or two protruding nucleotide sequences which are recognition sites for the same or different restriction endonucleases at opposite ends of the duplex.
Examples of protruding nucleotide sequences which are reco~nition sites for restriction enzymes include: 5' pA-A-T-T, the partial recognition site or EcoRI res-triction endonuclease; 5' pG-A-T-C, the partial recognition site for B mI res~riction endonuclease; and 5' pA-G-C-T, the paxtial recognitio~ site for HindIII restriction~endonuclease.
Similar adaptor molecules with partial recognition sites fox other restriction endonucleases such as PstI, SalI, HaeII, XmaI and ~II can also be synthesiæed and used or cloning.
The adaptor molecules of the inventio are prepared in a number of ways. First, duplex adaptor molecules having one protruding nucleotide sequence may be prepared by chemically synthesizing single-stranded oligonucleotides which include th~
recognition site for a restriction endonuclease and which are self-complementary. A double-stranded duplex is then prepared from the self-complementary oligonucleotides and the duplex is ~ ~1371 then contacted with the restriction endonuclease whose recognition site is included in the oligonucleotides.
The restriction endonuclease digests the duplex in such a way that an adaptor molecule having a protruding nucleo-tide seyuence which is the recognition site for the restriction endonuclease results.
For example, two self-complementary decadeoxyribo-nucleotides d(C-C-G-G-A-T-C-C-G-G) (BamI adaptor sequence including the BamI restriction endonuclease recognition site, 5' pG G-A-T-C-C) and d(A-C-A-A-G-C-T-T-G-T) (HindIII adaptor sequence including the IIindIII restriction endonuclease recognition site, 5' pA-A-G-C-T-T) were synthesized by the improved phosphotriester method developed previously ~Itakura, K. et al., Canadian J. Chem. 51, 3649 (1973~;
Itakura, K. et al., J. Am. Chem. Soc. 97, 7327 (1975);
Katagiri, N. et al., J. Am. Chem. Soc. 97, 7332 (1975);
Bahl, C.P., et al., Gene 1, 81 (1976); Stawinsky, J., et al., A Nucleic Acids Res. 4, ~ (1977)]~ Two new steps were intro-duced in these syntheses: the dimethoxytrityl group was removed by a 2% solution of benzenesulfonic acid in chloro-form and the chlorophenyl phosphate protecting group was removed by treatment with concentrated ammonium hydroxide for 4-6 hours. The oligonucleotides were characterized by two-dimensional electrophoresis-homochromatography of their partial venom phosphodiesterase digestion products ~Jay, E., et al., Nucleic Acids Res. 1, 331 (1974) and Tu, C.D., et al., A _ . Biochem 74, 7~ (1976)] which verified the above sequences of the t~lo synthetic decadeoxynucleotides.
~1~1137~
Double-stranded duple~es were prepared from these two self-complèmentary ol.igonucleotides. Specifically, the chemically synthesized decadeoxynucleotides (~00 pmoles) were phosphorylated at the 5' end using ~y32p] ATP and polynucleo-tide kinase [Wu, R. et al., Methods in Cancer Res., 12, 88 (1976)]. The labeled decadeoxynucleotides were dissolved in a suitable amount (about 100~1) of a suitable buffer such as 100 n~l Tris-HCl (pH 7.5) heated to about 90C. for about 1 minute, quickly chilled to about 0C. and then incubated at about 70C. for about 30 minutes. The duplexes of the deca-deoxynucleotides were forriled by slowly cooling the samples to about roorn temperature and then to about 4C.
The duplex which i.ncluded the recognition site for BamI restriction endonuclease was then contacted with this enzyme [Wilson, &.A. and Young, F.E., J. Mol. Biol. 97, 123 ~1975)] and after dlgestion gave an adaptor molecule having the protruding sequence 5' pG-A-T-C. Thus, 5' pC-C-G~G~A-T-C--C-G-G
3' G-G-C-C~-T-A-G-G-C-Cp ~ BamI endonuclease 5' C-C-G
3' G-G-C-C-T-A-Gp . A second method by which duplex adaptor molecules having one protruding nucleotide sequence may be prepared involves the chemical synthesis of a first single-stranded oli.gonucleotide which includes the recognition site for a restriction endonuclease and a second single-stranded oligonucleotide which does not include the recognition site for the restriction endonucléase but is otherwise complementary to the first, that is, one oligonucleotide is longer than the ~17~ 371 other and the nucleotides which correspond to the recognition site of the restriction endonuclease are protruding. Upon formin~ a double-stranded dupleY~ from the oligonucleotides, an adaptor molecule results in which the recognition site protrudes.
For example, an oligonucleotide having the sequence 5' pG-A-~-C-C-C-C-G-G-G can be synthesized by the method described previously, and similarly an oligonucleotide having the sequence 5' pC-C-C-G-G-G can be synthesized. A
duplex of these oligonucleotides can be formed as previously described which results in an adaptor molecule having the protruding sequence for BamI restriction endonuclease, namely, 5' pG-A-T-C. This type of adaptor can be defined as a ready-made adaptor. The following is illustrativeD
BamI site 5' pG-A-T-C-C-C-C-G-G-G
3' ~-G-5-C-C-C
Duplex adaptor molecules having two protruding nucleotide sequences which are recognition sites for the same or different restriction endonucleases can be prepared in a similar way. Single-stranded oligonucleotides which include the recognition site for a restriction endonuclease and are partially complementary are chemically synthesi~ed.
A double-stranded duplex is then formed from the oligonucleo-tides in which the nucleotide sequences which are recognition sites for the same or diffe~ent restriction endonucleases protrude at opposite ends of the duplex.
~ 17~ 3~
Conversion Adaptor Molecules Duplex conversion adaptor molecules having t~o pro-truding nucleotide sequences which are recognition sites for t~o different restriction endonucleases can be prepared by chemical synthesis of two single-stranded oligonucleotides which are partially complementary. A partially double-stranded duple~ is then formed from the ollgonucleotides in wllich the nucleotide sequences which are recognition sites for the two different restriction endonucleases protrude at opposite ends of the duplex~ The following example is illustrative:
BamI site 5' pG-A-T-C-C-C-C-G-G-G 3' G-G-G-C-C-C-T~T-A-Ap 5' ,t EcoRI site A conversion adaptor molecule with two different protruding nucleotide sequences can also be made by joining two dif~erent even-ended adaptor molecules using polynucleo-tide ligase, ~ollowed b~ digestion with the two appropriate restriction endonucleases~
A second type of conversion adaptor molecule, a completely single-stranded oligodeoxynucleotide, having two recognition sites for two different restriction endonucleases can be prepared in a similar way. This type of conversion adaptor molecule is used to convert a 3' protruding nucleo-tide sequence at the termini of a DNA molecule to be cloned to a 5' protruding sequence, or vice versa. For example, ~aeII restriction endonuclease digests DNA to give 3' protrud-ing ends, 3' d~C-G-C-G). In order to clone a DNA molecule such as DNA-X ~ith 3' protruding d(C-G-C-G) ends, the conversion adaptor can be joined to each end of DNA-X to produce a DNA
3 7 ~
molecule containing protruding 5' A-A-T-T ends. The latter can then be joined to the same 5' A-A-T-T ends of the cloning vehicle as follows:
DNA-X EcoRI site-HaeII conversion adaptor 5' G-C-G-C 3' 3' C-G-C-G S' pA-A-T-T-G-C-G-C
HaeII site EcoRI HaeII
- site site ~ T4 ligase 5' pA-A-T-T-G-C-G-C - -- G-C-G-C
3' 1 ~ C-G-C-G - C-G-C-G-T-T-A-Ap EcoRI site ~ ~
- EcoRI site cloning vehicle containing S' protruding pA-A-T-T sequence ~ ~ T4 ligase modified cloning vehicle .
In the event the DNA-X, after cloning, needs to be excised at the EcoRI or HaeII site, then the following conver-sion adaptor can he used:
5' pA-A-T-T-C-A-G-C-G-C
EcoRI site HaeII site Other examples of such conversion adaptors include: EcoRI
site - PstI site, BamI site - HaeII site, ~amI site - PstI
site, HindIII site - HaeII site, and HindIII site - Pst site.
~ daptor molecules of 3 different lengths can be prepared and used to adjust Ihe genetic informational material, e.g., DNA, ~hich is to be cloned to the proper reading frame for protein synthesis. Once the adaptor molecule is joined 1~7137~
to the genetic informational materia]. to be inserted and then inserted into a cloning vehicle and used to transform cells, the expression of the yenetic informational material in terms of RNA and protein synthesis depends upon whether the reading frame is correct. In cases where RNA synthesis is initiated rom a promoter site on the cloning vehicle, the number of nucleotides between the start of the mRNA and the beginning of the coding sequence of ,the DNA molecule should be a multiple of 3 in order to keep the reading frame for protein s~nthe.sis in phase during the translation of the mRNA sequence into protein sequence. If the exact length of genetic informational DNA is unknown, in order to make the length of genetic in~'orma.tional DNA plus the adaptor as multiples of 3, three different lengths of the adaptor (,3n, 3n+1 and 3n+2) must be available. Thus, all of the adaptors discussed must be constructed to give three different lengths. For example, if the adaptor 5' pG-A-T-C-C-C-C-G-G-G 3' G-G-G-C-C-C
is joined to genetic informational DNA to be cloned, it gives six extra nucleotides (multiple of 3, or 3n+0). The adaptor molecule can be converted to give 3n+1 and 3n+2 as follo~s:
5' pG-A-T-C-C-C-C-G-G-G 3' 5' pA-A-A-A 3' G-G-G-C-C-C + T-T-T-T
~ T4 ligase 5' pG-A-T-C-C-C-C-G-G-G-A-A-A-A 3' G-G-G-C-C-C-T-T-T-T
length 3n+1 I1~1371 If (pA)5:(pT)5 is used instead of (pA)~:(pT)4, then after joining it to ~he adaptor the duplex length will be 3n-~2.
In all the adaptors designed so ar, the DNA
sequence which corresponds to the termination codon for protein synthesis (U~, UAG, UGA) is avoided.
Adapted DNA Molecules Adapted DNA molecules can be prepared which include genetic informational material, e.g., DNA, typically in the form of a double-stranded duplex, which is to be inserted into a cloning vehicle and adaptor molecules joined to the ends of the genetic informational material. The genetic informational material can, within the limits imposed by the genetic code, be translated into any polypeptide. It can be naturally derived or synthetically produced.
The adaptor molecules described previously can be joined to opposite ends of genetic informational material which is to be inserted into a cloning vehicle using a poly-nucleotide ligase, e.g., T~ polynocleotide ligase or E. coli polynucleotide ligase. ~he adaptor molecules can be identical or different and can have one or two protruding nucleotide sequences depending upon the nature and structure of the genetic informational material such as DNA to which they are joined. The adaptor molecules are extremely useful tools in molecular clonlng since the same oligonucleotide adaptor molecules can serve to introduce any double-stranded DNA
molecule into cloning vehicles at speci-fic sites. The double-stranded DNA may be obtained by cleavage with a number o~
~12-1 ~137~
restriction endonucleases such as HaeIII and AluI to give even-ended DNA, or the ~uplex DNA may be chemically synthesized.
Alternatively, adapted DNA molecules can be pre-pared by chemically synthesizing a first single-stranded oligonucleotide which includes the genetic informational material to be inserted into a cloning vehicle and a recog-nition site for a restriction endonuc]ease and a second single-stranded oligonucleotide which is partially comple-mentary to the first oligonucleotide and includes a recognition site for a restriction endonuclease, either the same or a different endonuclease. A double-stranded duplex is then formed from the oligonucleotides in which the recognition sites protrude.
Still another method exists for preparing adapted DNA molecules. This involves chemically synthesizing self-complementary single-stranded oligonucIeotides which include the recognition site for a restriction endonuclease. Double-stranded duple~es are then formed from pairs of self-complementary oligonucleotides. The duplexes which are formed can be either identical or dif erent. They are enzymatically joined to opposite ends of the genetic informational matexial to be inserted into a cloning vehicle using a polynucleotide ligase. The resulting molecule is then digested with one or two restriction endonucleases depending upon whether the xecognition sites included in the duplexes were for identisal or different restric-tion endonucleases.
To illustrate this method of preparing an adapted D~A
molecule, the 21 nucleotide-long 12C operator dup]ex ~as .
3. ~137~
chemically synthesized. Two oligonucleotide duplexes were separately synthesized which contained the recognition site ~or a restriction endonuclease, in this case the site for BamI endonuclease. The latter duplexes were then joined to opposite ends of the lac operator using a polynucleotide ligase such as T4 ligase. The resulting molecule ~as then digested with BamI endonuclease which created protruding recognition sequences for BamI endonuclease, namely, 5' pG-A~T-C. The following depicts the reaction sequence:
5' pA-A-T-T-G-T-G-A-G-C-G-G-A-T-A-A-C-A-A-T-T
3' T-T-A-A-C-A-C-T-C-G-C-C-T-A-T-T-G T-T-A-Ap (lac operator) 5' pC-C-G-G-A-T-C-C-G-G
~2 3' G-G-C-C-T-A-G-G-C-Cp (decanucleotide duplex containing site ~ for BamI restriction endonuclease~
5' pC-C-G-G-A-T-C-C-G-G - C-C-G-G-A-m-C-C-G-G
(lac operator~
3' G-G-C-C-T-A-G-G-C-C - G-G-C-C-T-A-G-G-C-Cp BamI endonuclease 5' ~G-A-T-C-C-G-G - C-C-G
3' G-C-C -- G-G-C-C-T-A-Gp (adapted DNA molecule containing lac operator and protruding recognition sites for BamI endonuclease) Alternatively, a lac operator DNA has also been synthesized which included the protruding recognition sites ~or EcoRI éndonuclease [Marians, K.J. et al., Nature ~3, 744 (1976)]. This lac operator DNA can be joined directly to the DNA to be cloned at the EcoRI site, and then to the cloning vehicle.
~17137~
To m~re specifically illustrate, an adap-ted DNA
molecule which included lac operator DNA and protruding nucleotide sequences at both ends which were recot~nition sites for BamI endonuclease was prepared as follows: First, the synthetic decadeoxyribonucleotide duplex which included the recognition site for _ mI endonuclease was prepared as described previously. The 21 nucleotide-long synthetic lac operator was prepared. [Bahl, C.P. et al., PNAS 73, 91 (1976~] ~hen the synthetic duplex (about 10 pmole) and the synthetic lac operator (about 1.0 pmole) were joined end-to-end [Sgaramella, V. et al., PNAS 67, 1468 (1970)~ by incubating with about 3 units of T~ DNA ligase in about 50 ~1 of a solu-tion containing about 20~M Tris-~lCl (pH 7.5), about lOmM dithio-threitol, about lOmM MgC12 and about 35 ~M ~TP at about 20C.
for about 6 hours. The solution was heated to about 70C. for about 5 minutes to inactivate the ligase and cooled slowly to room temperature, 2 volumes of ethanol were added, and after about 12 hours at about 20~C the DNA was pelleted at about 10,000 ~ or about 1 hour. The pellet was dissolved in about 50 ~1 of a solution containing about 6.6mM Tri~-HCl (pH 7.5~ t about 6.6mM ~gC12 and about lmM dithiothreitol~ To this solu-tion was added 2 units of BamI endonuclease. The sample was incubated at about 37C. for about 12 hours to produce the adapted DNA molecule.
Modified Cloning Vehicles _ _ Modified cloning vehicles can be prepared from a cloning vehicle such as plasmid, phage or viral DNA which has been contacted with a restriction endonuclease so that a pro-truding oligonucleotide complementary to the recognition site -1 1 7 :1 3 ~
for the restriction enzyme has resulted by joining to such an endonuclease-treated cloning vehicle an adapted DNA
molecule such as has been described hereinabove. Specific cloning vehicles include p~lB9 plasmid DNA and ~ phage DNA.
The adapted DNA molecule and the endonuclease-treated cloning vehicle can be enzyma~ically joined by use of a polynucleotide ligase such as T4 polynucleotide ligase or E. coli. polynucleo-tide ligase. If the restriction endonuclease site at the termini of the cloning vehicle is different from that of the DNA molecule to be cloned, either the cloning vehicle or the DNA to be cloned can be modified by the use of adaptors.
By this method, synthetic lac operator DNA has been inserted at the BamI, HindIII and EcoRI sites o pMB9 DNA.
For example, linear pMB9 DNA prepared by contacting pMB9 DNA
with BamI restriction endonuclease and an adapted DNA molecule which included lac operator DNA and protruding nucleotide sequences at both ends which were recognition sites or BamI
endonuclease were heated to about 70C. for about 5 minutes~
cooled slowly to room temperature, and the DNA was precipitated by adding 2 volumes of ethanol and after about 12 hours at about 20C. the DNA was pelleted at about 10,000 g for about 1 hour.
The DNA pellet was dissolved in about 50 ~1 of a solution contain-ing about 20mM Tris-HC1 (pH 7.5), about lOmM MgC12, about lOmM
dithiothreitol and about 35 ~M ATP. Three units of DNA ligase ere added and the samples were incubated at about 12.5C. for ahout 24 hours to produce lac-p~9 DNA. After heating at about 70C. for about 5 minutes, and slowly cooling to roo~ tempera-ture, the lac-pMB9 DNA was used directly for transformation as described below.
~17~ ~7~
This method is also suitable for the insertion of mul-tiple copies of a gene such as lac opera~or DNA into a cloning vehicle or for the insertion of a combination of genes.
In order to characterize the lac-pMB9 DNA, it was isolated after transformation and amplification by the addi-tion of chloramphenicol to the bacterial culture, labelled by nick translation [Maniatis, T. et al., _NAS 72~ 1184 (1975)]
and then studied for lac repressor binding properties. The inhibitory effect of isopropyl thioglactoside (IPTG) showed that the binding was specific, thus confirming that a lac operator had been inserted into the plasmid.
The modified plasmid DNA was further characterized by digestion wi~h the appropriate restriction endonuclease and then labelled at the 3'-ends by repair synthesis in the presence of [~-3 P~ dNTP [Wu, ~. et al., Methods in Cancer ~esearch 12, 88 (1976)]. This gave two fragments on poly-acrylamide gel electrophoresis which corresponded to the linear pMB9 DNA and to the lac operator.
Transformation The transfer of genetic informational material such as DNA into host cells, e.g., E. coli cells, can be effected by transformation using the modified cloning vehicles. For example, the lac p~lB9 DNA was used to transform competent E. coli HB 129 cells as follows: The D~A and r~cipient cells -~ere mixed together and incubated at about 0C. for about 30 minutes. The temperature of the mixture was raised to about ~2C. for about 2 minutes and thenchilled to facilitate upta~e ~ ~137~
of the DNA by the cells. ~bout nine volumes of pre~larmed L-broth were added and the cells allowed to recover at about 37C. for about 2 hours. One volume of L-broth supplemented with about 10 ~g ml l of tetracycline was then added. After an additional approximately 30 minutes at 37C., the tetracycline concentration was brought up to a final level of about 20 ~g ml 1 This cell suspension was used to inoculate 100 ml of M9 medium for the isolation of a larger amount of plasmid DNA [Katz, L. and Helinski, D . R., J . Bact.
119, 450 (1973)]. This plasmid DNA (about 30 ~g) which con-tained some modified lac-pMB9 D~A was enriched for lac sequences by binding it to the lac repressor (about 4.5 ~g) on ~ ~illipore filter and eluting it with about l ml of lmM
~ h/~g~/ac7~osld~
isopropyl thioglactocido (IPTG). This DNA (about 6% of the input), enriched for lac sequences, was used for a subsequent transformation on nutrient agar plates containing about 20 ~g ml of tetracycline~ The frequency of transformation was 6.4 x 10 transformants per ~g of recombinant DNA per viable cell, whereas in the same conditions native pMB9 gave a fre-quency of 1.8 x 10 2 Since about l x 107 viable cells were used, the number of transEormants per ~g of recombinant DNA
was about 6,000.
~lethod for Determining Whether Genetic Informational Material Such as DNA Has Been Transferred In order to determine whether ~enetic informational DNA has been transferred into host cells, an indicator DNA
molecule such as the lac operator can be included as part of the yenetic informational DNA in an adapted D~A molecule.
This cdapted DNA molecule is then inserted into a cloning vehicle to prepare a modified cloning vehicle and host cells are transformed using the modified cloning vehicle. If the ~7137~
lae operator is used, transformed cells are sereened to determine whether transfer has taken plaee by platlng the transformed eells in agar plates containing x-gal which will result in the produetion of blue colonies i~ the genetie informational DNA has been transferred because transformed eells which include the lac operator will be constitutive ~or ~-galactosidase synthesis [Miller, J.H., Experiments in Molecular Geneties, Cold Spring ~arbor Lab. 48 (1972)].
. . _ _ .
As will be obvious to one skilled in the art, many modifications in the invention are possible ~ithout departing from the spirit and seope thereof.
~19-
Claims (50)
1. An adaptor molecule for insertion of genetic informational material into a cloning vehicle which comprise an oligonucleotide selected from:
(a) a double-stranded oligonucleotide having at least one protruding nucleotide sequence which is a recognition site for a restriction endonuclease, and (b) a single-stranded oligonucleotide having a nucleotide sequence which includes the recognition site for two different restriction endonucleases at opposite ends of the oligonucleotide.
(a) a double-stranded oligonucleotide having at least one protruding nucleotide sequence which is a recognition site for a restriction endonuclease, and (b) a single-stranded oligonucleotide having a nucleotide sequence which includes the recognition site for two different restriction endonucleases at opposite ends of the oligonucleotide.
2. An adaptor molecule of Claim 1 wherein the protruding nucleotide sequence is a part of the recognition site for EcoRI endonuclease.
3. An adaptor molecule of Claim 1 wherein the protruding nucleotide sequence is a part of the recognition site for BamI endonuclease.
4. An adaptor molecule of Claim 1 wherein the protruding nucleotide sequence is a part of the recognition site for HindIII endonuclease.
5. An adaptor molecule of Claim 1 wherein the protruding nucleotide sequence is part of the recognition site for PstI endonuclease.
6. An adaptor molecule of Claim 1 wherein the protruding nucleotide sequence is a part of the recognition site for SalI endonuclease
7. An adaptor molecule of Claim 1 wherein the protruding nucleotide sequence is a part of the recognition site for HaeII endonuclease.
8. An adaptor molecule of Claim 1 wherein the protruding nucleotide sequence is a part of the recognition site for XmaI endonuclease.
CLAIMS:
CLAIMS:
9. An adaptor molecule of Claim 1 wherein the pro-truding nucleotide sequence is a part of the recognition site for BglII endonuclease.
10. An adaptor molecule as in claim 1 for insertion of genetic informational material into a cloning vehicle which comprises a double-stranded oligonucleotide having first and second protruding nucleotide sequences which are recognition sites for restriction endonucleases.
11. The adaptor molecule of Claim 10 wherein said first and second protruding nucleotide sequences are recognition sites for the same restriction endonuclease.
12. The adaptor molecule of Claim 10 wherein said first and second protruding nucleotide sequences are recognition sites for different restriction endonucleases.
13. An adaptor molecule of Claim 1 for insertion of genetic informational material into a cloning vehicle which comprises a single-stranded oligonucleotide having a nucleo-tide sequence which includes the recognition sites for two different restriction endonucleases at opposite ends of the oligonucleotide.
14. A method of preparing the adaptor molecule of Claim 1 (a) which comprises chemically synthesizing single-stranded oligonucleotides which include the recognition site for a restriction endonuclease and which are self comple-mentary, forming a double-stranded duplex from the self-complementary oligonucleotides and digesting the duplex with the restriction endonuclease whose recognition site is in-cluded in the duplex so that the adaptor molecule results.
15. A method of preparing the adaptor molecule of Claim 1 (a) which comprises chemically synthesizing a first CLAIMS:
15. A method of preparing the adaptor molecule of Claim 1 (a) which comprises chemically synthesizing a first CLAIMS:
15. cont.
single-stranded oligonucleotide which includes the recogni-tion site for a restriction endonuclease, chemically synthe-sizing a second single-stranded oligonucleotide which does not include the recognition site for the restriction endo-nuclease but is otherwise complementary to said first oligo-nucleotide, and forming a double-stranded duplex from the oligonucleotides in which the recognition site for the rest-riction endonuclease protrudes.
single-stranded oligonucleotide which includes the recogni-tion site for a restriction endonuclease, chemically synthe-sizing a second single-stranded oligonucleotide which does not include the recognition site for the restriction endo-nuclease but is otherwise complementary to said first oligo-nucleotide, and forming a double-stranded duplex from the oligonucleotides in which the recognition site for the rest-riction endonuclease protrudes.
16. A method of preparing the adaptor molecule of Claim 10 which comprises chemically synthesizing a first single-stranded oligonucleotide which includes a first recognition site for a first restriction endonculease, chemically synthesizing a second single-stranded oligo-nucleotide which includes a second recognition site for a second restriction endonuclease and is partially complementary to said first oligonucleotide, and forming a double-stranded duplex from the oligonucleotides in which said first and second recognition sites protrude.
17. Adaptor molecules of Claim 1 joined to each end of DNA which comprises genetic informational material to be in-serted into a cloning vehicle.
18. The adapted DNA molecule of Claim 17 wherein the genetic informational material to be inserted is a double-stranded duplex.
19. The adapted DNA molecule of Claim 18 wherein the genetic informational material to be inserted comprises the lac operator
20. The adapted DNA molecule of Claim 17 wherein said adaptor molecules are those of Claim 1 (a).
21. The adapted DNA molecule of Claim 17 wherein said adaptor molecules are those of Claim 10.
CLAIMS:
CLAIMS:
22. The adapted DNA molecule of Claim 17 wherein one adaptor molecule is an adaptor molecule of Claim 1 (a) and the other adaptor moleucle is an adaptor molecule of Claim 10.
23. The adapted DNA molecule of Claim 17 wherein one said adaptor molecule is an adaptor molecule of Claim 11 and the other adaptor molecule is an adaptor molecule of Claim 1(a).
24. The method of Claim 14 including subsequently enzymatically joining said adaptor molecules to ends of DNA including genetic informational material to be inserted into a cloning vehicle, using a polynucleotide ligase.
25. The method of Claim 15 including subsequently enzymatically joining said adaptor molecules to ends of DNA including genetic informational material to be inserted into a cloning vehicle, using a polynucleotide ligase.
26. The method of Claim 16 including subsequently enzymatically joining said adaptor moleucles to ends of DNA including genetic informational material to be inserted into a cloning vehicle, using a polynucleotide ligase.
27. The method of Claim 24, 25 or 26 wherein the polynucleotide ligase is T4 polynucleotide ligase.
28. The method of Claim 24, 25 or 26 wherein the polynucleotide ligase is E. coli polynucleotide ligase.
29. The method of Claim 26 wherein one of the adaptor molecules on each DNA molecule is a double-stranded oligo-nucleotide having a single protruding recognition site.
30. The method of Claim 24 or 25 wherein one of the adaptor molecules on each DNA molecule is a double-stranded oligonucleotide having first and second protruding recognition sites.
CLAIMS:
CLAIMS:
31. A method of preparing the adapted DNA molecule of Claim 17 which comprises chemically synthesizing a first single-stranded oligonucleotide which includes the genetic informational material to be inserted into the cloning vehicle and a first recognition site for a first restriction endonuclease, chemically synthesizing a second single-stranded oligonucleotide which is partially complementary to said first oligonucleotide and includes a second recognition site for a second restriction endonuclease and forming a duplex from the oligonucleotides in which said first and second recognition sites protrude.
32. The method of Claim 31 wherein said first and said second recognition sites are for the same restriction endonuclease.
33. The method of Claim 31 wherein said first and second recognition sites are for different restriction endonucleases.
34. A method of preparing the adapted DNA molecule of Claim 17 which comprises chemically synthesizing a first self-complementary single-stranded oligonucleotide which includes the recognition site for a first restriction endo-nuclease and a second self-complementary single-stranded oligonucleotide which includes the recognition site for a second restriction endonuclease, forming a first double-stranded duplex from a pair of said first oligonucleotides and a second double-stranded duplex from a pair of said second oligonucleotides; enzymatically joining said first and second duplexes to opposite ends of the genetic infor-mation material to be inserted into a cloning vehicle using a polynucleotide ligase; and digesting the resulting CLAIMS:
34. A method of preparing the adapted DNA molecule of Claim 17 which comprises chemically synthesizing a first self-complementary single-stranded oligonucleotide which includes the recognition site for a first restriction endo-nuclease and a second self-complementary single-stranded oligonucleotide which includes the recognition site for a second restriction endonuclease, forming a first double-stranded duplex from a pair of said first oligonucleotides and a second double-stranded duplex from a pair of said second oligonucleotides; enzymatically joining said first and second duplexes to opposite ends of the genetic infor-mation material to be inserted into a cloning vehicle using a polynucleotide ligase; and digesting the resulting CLAIMS:
34. cont.
molecule with said first and second restriction endo-nucleases so that protruding recognition sites for said first and second restriction endonculeases result.
molecule with said first and second restriction endo-nucleases so that protruding recognition sites for said first and second restriction endonculeases result.
35. The method of Claim 34 wherein said first and second restriction endonucleases are identical.
36. The method of Claim 34 wherein said first and second restriction endonucleases are different.
37. The adapted DNA molecule of Claim 17, joined to a cloning vehicle which had been contacted with a restriction endonuclease so that a protruding oligonucleotide comple-mentary to the recognition site for said restriction endo-nuclease resulted.
38. The modified cloning vehicle molecule of Claim 37 wherein the cloning vehicle is plasmid DNA.
39. The modified cloning vehicle of Claim 37 wherein the cloning vehicle is phage DNA.
40. The modifed cloning vehicle of Claim 37 wherein the cloning vehicle is viral DNA.
41. The modified cloning vehicle of Claim 38 wherein the cloning vehicle is pMB9 DNA.
42. The modified cloning vehicle of Claim 39 wherein the cloning vehicle is A phage DNA.
43. A method of preparing the combination of Claim 37 which comprises contacting a cloning vehicle with a rest-riction endonuclease so that a protruding oligonucleotide complementary to the recognition site for said restriction endonuclease results, and joining the adapted DWA molecule which includes the recognition site for said restriction endonuclease with said cloning vehicle using a polynucleo-tide ligase.
CLAIMS:
CLAIMS:
44. A method of transferring genetic informational material into hosts cells which comprises transformation of said host cells with the adapted DNA-cloning vehicle combi-nation of Claim 37.
45. A modified cloning vehicle which comprises a cloning vehicle and an adapted DNA molecule in accordance with Claim 17.
46. A modified cloning vehicle in accordance with Claim 45 wherein said cloning vehicle is plasmid DNA.
47. A modified cloning vehicle in accordance with Claim 45 wherein said cloning vehicle is phage DNA.
48. A modified cloning vehcile in accordance with Claim 45 wherein said cloning vehicle is viral DNA.
49. A modified cloning vehicle in accordance with Claim 45 wherein said cloning vehicle is pMB9 DNA.
50. A modified cloning vehicle in accordance with Claim 45 wherein said cloning vehicle is .lambda. phage DNA.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US05/843,422 US4321365A (en) | 1977-10-19 | 1977-10-19 | Oligonucleotides useful as adaptors in DNA cloning, adapted DNA molecules, and methods of preparing adaptors and adapted molecules |
| US843,422 | 1977-10-19 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1171371A true CA1171371A (en) | 1984-07-24 |
Family
ID=25289929
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000313650A Expired CA1171371A (en) | 1977-10-19 | 1978-10-18 | Oligonucleotides useful as adaptors and adapted molecules for cloning dna and method of employing same |
Country Status (2)
| Country | Link |
|---|---|
| US (1) | US4321365A (en) |
| CA (1) | CA1171371A (en) |
Families Citing this family (56)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5221619A (en) * | 1977-11-08 | 1993-06-22 | Genentech, Inc. | Method and means for microbial polypeptide expression |
| US5583013A (en) * | 1977-11-08 | 1996-12-10 | Genentech, Inc. | Method and means for microbial polypeptide expression |
| US4342832A (en) * | 1979-07-05 | 1982-08-03 | Genentech, Inc. | Method of constructing a replicable cloning vehicle having quasi-synthetic genes |
| US4769326A (en) * | 1980-02-29 | 1988-09-06 | The Regents Of The University Of California | Expression linkers |
| US4617384A (en) * | 1980-03-27 | 1986-10-14 | Cornell Research Foundation, Inc. | Adaptor molecules for DNA and their application to synthesis of gene-derived products |
| US4401759A (en) * | 1980-05-01 | 1983-08-30 | Harvey Rubin | Detection and isolation of glucagon mRNA using a synthetic oligodeoxynucleotide |
| IE52417B1 (en) * | 1980-12-12 | 1987-10-28 | Unilever Plc | Dna sequences encoding various allelic forms of mature thaumatin,recombinant plasmids comprising said dnas and a process for their preparation,bacterial cultures comprising said recombinant plasmids,and method for producing mature thaumatin |
| DE3177281T2 (en) * | 1980-12-12 | 1992-12-10 | Unilever Nv | STRUCTURAL GENES ENCODING THE DIFFERENT ALLELIC AND RIPENING FORMS OF PRAEPROTHAUMATIN AND THEIR MUTANTS, THESE CLONING VECTORS CONTAINING THESE STRUCTURAL GENES AND THEIR EXPRESSION IN MICROBIAL CELLS. |
| US4792602A (en) * | 1981-06-19 | 1988-12-20 | Cornell Research Foundation, Inc. | Adaptors, and synthesis and cloning of proinsulin genes |
| JP2547714B2 (en) * | 1981-10-23 | 1996-10-23 | モルキユラ− バイオシステムズ インコ−ポレテツド | Oligonucleotide therapeutic agent and method for producing the same |
| US5252482A (en) * | 1982-05-20 | 1993-10-12 | Suntory Limited | Precursor of a C-terminal amidated calcitonin |
| EP0118551A1 (en) * | 1982-09-15 | 1984-09-19 | Collaborative Research Inc. | Production of interferon in yeast by use of invertase promoter |
| US4547569A (en) * | 1982-11-24 | 1985-10-15 | The United States Of America As Represented By The Department Of Health And Human Services | Intercalating agents specifying nucleotides |
| FR2540122B1 (en) * | 1983-01-27 | 1985-11-29 | Centre Nat Rech Scient | NOVEL COMPOUNDS COMPRISING A SEQUENCE OF OLIGONUCLEOTIDE LINKED TO AN INTERCALATION AGENT, THEIR SYNTHESIS PROCESS AND THEIR APPLICATION |
| US4661450A (en) * | 1983-05-03 | 1987-04-28 | Molecular Genetics Research And Development Limited Partnership | Molecular cloning of RNA using RNA ligase and synthetic oligonucleotides |
| DE3326520A1 (en) * | 1983-07-22 | 1985-01-31 | Hubert Prof. Dr. 2000 Hamburg Köster | METHOD FOR DEFINED LINKING OF DOUBLE-HELICAL DNA FRAGMENTS BY MEANS OF A LINKER DNA AND OLIGONUCLETIDES SUITABLE AS LINKER DNA |
| US4582802A (en) * | 1983-09-30 | 1986-04-15 | The United States Of America As Represented By The Department Of Health And Human Services | Stimulation of enzymatic ligation of DNA by high concentrations of nonspecific polymers |
| EP0177497A4 (en) * | 1984-04-05 | 1987-07-06 | Life Technologies Inc | Immobilization of nucleic acids. |
| EP0175689B1 (en) * | 1984-04-06 | 1992-07-22 | Life Technologies Inc. | Method of detecting nucleic acid sequences |
| DE3507881A1 (en) * | 1985-03-06 | 1986-09-11 | Hoechst Ag, 6230 Frankfurt | METHOD AND MEANS FOR PHOSPHORYLATION |
| DE3588239T3 (en) * | 1985-03-30 | 2007-03-08 | Kauffman, Stuart A., Santa Fe | A method for obtaining DNA, RNA, peptides, polypeptides or proteins by DMS recombinant methods |
| JP2609238B2 (en) * | 1985-05-02 | 1997-05-14 | ジエネテイツクス・インスチチユ−ト・インコ−ポレ−テツド | Methods and nucleic acid constructs for making reagent complexes useful for assaying a target nucleotide sequence |
| US4937189A (en) * | 1985-10-18 | 1990-06-26 | Pfizer Inc. | Expression and secretion of heterologous proteins by Yarrowia lipolytica transformants |
| US5085982A (en) * | 1986-06-12 | 1992-02-04 | City Of Hope | Method of detecting specific substances by selective growth of living cells |
| US20060008806A1 (en) * | 1986-07-17 | 2006-01-12 | University Of Washington | Method for producing novel DNA sequence with biological activity |
| US5082767A (en) * | 1989-02-27 | 1992-01-21 | Hatfield G Wesley | Codon pair utilization |
| US5599696A (en) * | 1989-10-17 | 1997-02-04 | California Institute Of Technology | Method of preparing nucleic acids having an undefined nucleotide sequence amplification |
| US7026115B1 (en) * | 1991-09-24 | 2006-04-11 | Keygene N.V. | Selective restriction fragment amplification: fingerprinting |
| DK0969102T3 (en) * | 1991-09-24 | 2008-05-13 | Keygene Nv | Primers, kits and sets of restriction fragments used in selective restriction fragment amplification |
| US20100267023A1 (en) * | 1992-09-24 | 2010-10-21 | Keygene N.V. | Selective restriction fragment amplification: fingerprinting |
| US5663062A (en) * | 1992-04-03 | 1997-09-02 | Stratagene | Oligonucleotide libraries useful for producing primers |
| US5552278A (en) * | 1994-04-04 | 1996-09-03 | Spectragen, Inc. | DNA sequencing by stepwise ligation and cleavage |
| US5831065A (en) * | 1994-04-04 | 1998-11-03 | Lynx Therapeutics, Inc. | Kits for DNA sequencing by stepwise ligation and cleavage |
| US5714330A (en) * | 1994-04-04 | 1998-02-03 | Lynx Therapeutics, Inc. | DNA sequencing by stepwise ligation and cleavage |
| US6013445A (en) * | 1996-06-06 | 2000-01-11 | Lynx Therapeutics, Inc. | Massively parallel signature sequencing by ligation of encoded adaptors |
| USRE43097E1 (en) | 1994-10-13 | 2012-01-10 | Illumina, Inc. | Massively parallel signature sequencing by ligation of encoded adaptors |
| US20040266706A1 (en) * | 2002-11-05 | 2004-12-30 | Muthiah Manoharan | Cross-linked oligomeric compounds and their use in gene modulation |
| US6060245A (en) * | 1996-12-13 | 2000-05-09 | Stratagene | Methods and adaptors for generating specific nucleic acid populations |
| US6017701A (en) * | 1996-12-13 | 2000-01-25 | Stratgene | Methods and adaptors for generating specific nucleic acid populations |
| US20020177138A1 (en) * | 2000-11-15 | 2002-11-28 | The United States Of America , Represented By The Secretary, Department Of Health And Human Services | Methods for the indentification of textual and physical structured query fragments for the analysis of textual and biopolymer information |
| AUPR446801A0 (en) | 2001-04-18 | 2001-05-17 | University Of Queensland, The | Novel compositions and uses therefor |
| US20040224330A1 (en) * | 2003-01-15 | 2004-11-11 | Liyan He | Nucleic acid indexing |
| US7365179B2 (en) * | 2003-09-09 | 2008-04-29 | Compass Genetics, Llc | Multiplexed analytical platform |
| WO2005080604A2 (en) | 2004-02-12 | 2005-09-01 | Compass Genetics, Llc | Genetic analysis by sequence-specific sorting |
| US7407757B2 (en) * | 2005-02-10 | 2008-08-05 | Population Genetics Technologies | Genetic analysis by sequence-specific sorting |
| EP1856293A2 (en) * | 2005-03-16 | 2007-11-21 | Compass Genetics, Llc | Methods and compositions for assay readouts on multiple analytical platforms |
| WO2007100384A2 (en) * | 2005-10-24 | 2007-09-07 | Invitrogen Corporation | Three frame cdna expression libraries for functional screening of proteins |
| US20070231906A1 (en) * | 2005-10-25 | 2007-10-04 | Invitrogen Corporation | Three Frame cDNA Expression Libraries for Functional Screening of Proteins |
| US9593340B2 (en) * | 2007-10-15 | 2017-03-14 | Admedus Vaccines Pty Ltd. | Expression system for modulating an immune response |
| US8017328B2 (en) * | 2008-11-21 | 2011-09-13 | Agilent Technologies, Inc. | Genome partitioning using a nicking endonuclease |
| US9795658B2 (en) | 2010-04-20 | 2017-10-24 | Admedus Vaccines Pty Ltd | Expression system for modulating an immune response |
| US9057734B2 (en) | 2010-08-23 | 2015-06-16 | President And Fellows Of Harvard College | Optogenetic probes for measuring membrane potential |
| JP2015505979A (en) | 2011-11-23 | 2015-02-26 | プレジデント アンド フェローズ オブ ハーバード カレッジ | System and method for imaging with high spatial and / or temporal accuracy |
| AU2016367712B2 (en) | 2015-12-09 | 2021-10-07 | Jingang Medicine (Australia) Pty Ltd | Immunomodulating composition for treatment |
| US11285191B2 (en) | 2016-07-26 | 2022-03-29 | The Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Immunostimulatory compositions and uses therefor |
| WO2023049826A1 (en) | 2021-09-23 | 2023-03-30 | President And Fellows Of Harvard College | Genetically encoded voltage indicators and uses thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4237224A (en) * | 1974-11-04 | 1980-12-02 | Board Of Trustees Of The Leland Stanford Jr. University | Process for producing biologically functional molecular chimeras |
-
1977
- 1977-10-19 US US05/843,422 patent/US4321365A/en not_active Expired - Lifetime
-
1978
- 1978-10-18 CA CA000313650A patent/CA1171371A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| US4321365A (en) | 1982-03-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1171371A (en) | Oligonucleotides useful as adaptors and adapted molecules for cloning dna and method of employing same | |
| US4661450A (en) | Molecular cloning of RNA using RNA ligase and synthetic oligonucleotides | |
| US4719179A (en) | Six base oligonucleotide linkers and methods for their use | |
| US6277632B1 (en) | Method and kits for preparing multicomponent nucleic acid constructs | |
| Lathe et al. | Linker tailing: unphosphorylated linker oligonucleotides for joining DNA termini | |
| US5223414A (en) | Process for nucleic acid hybridization and amplification | |
| JP2547714B2 (en) | Oligonucleotide therapeutic agent and method for producing the same | |
| US5279952A (en) | PCR-based strategy of constructing chimeric DNA molecules | |
| JP6817493B2 (en) | Method for producing single-strand RNA | |
| CN107250148A (en) | Guide RNA with chemical modification | |
| JP2022530432A (en) | Nucleic acid construct and its production method | |
| Rothstein et al. | [7] Synthetic adaptors for cloning DNA | |
| Zuckermann et al. | Site-selective cleavage of structured RNA by a staphylococcal nuclease-DNA hybrid. | |
| WO1985004674A1 (en) | Immobilization of nucleic acids | |
| WO2022227880A1 (en) | Novel phosphorylated adenylase, and preparation method therefor and application thereof | |
| JPH0343085A (en) | Method and composition for multiplying and detecting sequence of nucleic acid | |
| El-Sagheer et al. | Single tube gene synthesis by phosphoramidate chemical ligation | |
| US5523221A (en) | Method for the directional cloning of DNA | |
| CA2197420A1 (en) | Construction of nucleoprotein based assemblies comprising addressable components for nanoscale assembly and nanoprocessors | |
| JP2023514422A (en) | Methods and Products for Producing Single-Stranded DNA Polynucleotides | |
| CA2205082C (en) | Method of forming double stranded dna polymer having repeating subunits in tandem | |
| WO1988005082A1 (en) | Microbial production of peptide oligomers | |
| JPH03504797A (en) | gene synthesis | |
| Yang et al. | Directional cloning of an oligonucleotide fragment into a single restriction site | |
| Chapman et al. | Secondary lambda attachment site in the threonine operon attenuator of Escherichia coli |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |