CA2375027A1 - High specificity primers, amplification methods and kits - Google Patents

High specificity primers, amplification methods and kits Download PDF

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Publication number
CA2375027A1
CA2375027A1 CA002375027A CA2375027A CA2375027A1 CA 2375027 A1 CA2375027 A1 CA 2375027A1 CA 002375027 A CA002375027 A CA 002375027A CA 2375027 A CA2375027 A CA 2375027A CA 2375027 A1 CA2375027 A1 CA 2375027A1
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Prior art keywords
primer
loop
nucleotides
complementary
sequence
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CA002375027A
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French (fr)
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CA2375027C (en
Inventor
Sanjay Tyagi
Fred R. Kramer
Robert Vartikian
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Rutgers State University of New Jersey
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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Abstract

For nucleic acid amplification including extension of primers by a DNA polymerase, high specificity primers are provided. The primers include a typ e of hairpin structure in which a single-stranded loop separates complementary 3' and 5' arms and in which the loop and the 3' arm are complementary to the target nucleic acid. Amplification methods, assays and kits including such primers are included in the invention.

Claims (41)

1. An hairpin oligonucleotide primer for extension by a DNA
polymerase comprising a stem formed by complementary 3' and 5' arm sequences and a single-stranded loop sequence separating said arm sequences, wherein said 3' arm sequence and said loop sequence are both perfectly complementary to a selected priming region of a target nucleic acid strand and wherein hybridization of the loop sequence to a model non-target sequence of the length of the loop and perfectly complementary to the loop sequence does not cause dissociation of the stem.
2. The hairpin primer according to claim 1 additionally comprising interactive fluorescent label moieties attached to the 3' and 5' arm sequences, whereby incorporation of the primer into a double-stranded nucleic acid detestably alters the fluorescence emitted by said label moieties.
3. The hairpin primer according to claim 2 wherein said interactive fluorescent label moieties comprise a fluorophore and a non-fluorescent quencher.
4. The hairpin primer according to claim 1 wherein said loop sequence is 5 to 9 nucleotides in length and said complementary 3' and 5' arm sequences form a stem that is 5 to 12 nucleotides in length.
5. The hairpin primer according to claim 4 wherein said priming region contains a single nucleotide subject to mutation and said loop sequence is complementary to the portion of said priming region containing said nucleotide.
6. The hairpin primer according to claim 4 wherein said primer contains a terminator nucleotide between the loop sequence and the 5' arm sequence.
7. The hairpin primer according to claim 4 containing modified nucleotides, modified internucleotide linkages, or both.
8. The hairpin primer according to claim 4 additionally comprising interactive fluorescent label moieties attached to the 3' and 5' arm sequences, whereby incorporation of the primer into a double-stranded nucleic acid detectably alters the fluorescence emitted by said label moieties.
9. The hairpin primer according to claim 6 wherein said interactive fluorescent label moieties comprise a fluorophore and a non-fluorescent quencher.
10. The hairpin primer according to claim 1 containing modified nucleotides, modified internucleotide linkages, or both.
11. The hairpin primer according to claim 1 wherein said primer contains a terminator nucleotide between the loop sequence and the 5' arm sequence.
12. The hairpin primer according to claim 1 wherein said priming region contains a single nucleotide subject to mutation and said loop sequence is complementary to the portion of said priming region containing said nucleotide.
13. The hairpin primer according to claim 12 additionally comprising interactive fluorescent label moieties attached to the 3' and 5' arm sequences, whereby incorporation of the primer into a double-stranded nucleic acid detectably alters the fluorescence emitted by said label moieties.
14. The hairpin primer according to claim 13 wherein said interactive fluorescent label moieties comprise a fluorophore and a non-fluorescent quencher.
15. In a linear oligonucleotide primer for extension by a DNA
polymerase, said primer having a 5' terminus and a 3' terminal region, the improvement comprising adding to said 5' terminus a nucleotide sequence that is complementary to said 3' terminal region to form an hairpin structure comprising a stem and loop, wherein hybridization of the loop to a model oligonucleotide having the same length as the loop and being perfectly complementary thereto does not cause said stem to dissociate.
16. The improvement according to claim 15 wherein said loop is 5 to 9 nucleotides in length and said stem is 5 to 12 nucleotides in length.
17. The improvement according to claim 15 further comprising adding a terminator nucleotide to the 5' terminus prior to adding said nucleotide sequence that is complementary to said 3' terminal region.
18. In a process for nucleic acid amplification comprising the extension of an oligonucleotide primer by a DNA polymerase, said primer having a 5' terminus and a 3' terminal region, the improvement comprising adding to said 5' terminus a nucleotide sequence that is complementary to said 3' terminal region to form an hairpin structure comprising a stem and loop, wherein hybridization of the loop to a model oligonucleotide having the same length as the loop and being perfectly complementary thereto does not cause said stem to dissociate.
19. The improvement according to claim 18 wherein said loop is 5 to 9 nucleotides in length and said stem is 5 to 12 nucleotides in length.
20. A process for nucleic acid amplification comprising the extension by a DNA polymerase of at least one oligonucleotide primer according to claim 1.
21. The process according to claim 20 selected from the group consisting of a polymerase chain reaction (PCR), a strand displacement reaction (SDA), a nucleic acid sequence-based amplification (NASBA), a transcription-mediated amplification (TMA), and a rolling-circle amplification (RCA).
22. The process according to claim 21 wherein said at least one primer according to claim 1 comprises a pair of amplification primers.
23. The process according to claim 21 wherein said at least one primer according to claim 1 includes a terminator nucleotide between the loop and the 5' arm.
24. The process according to claim 20 including real-time detection of intended amplification products utilizing separate detector probes having interactive labels, at least one of which is a fluorophore.
25. The process according to claim 20 including real-time detection, wherein said at least one primer additionally comprises interactive fluorescent label moieties attached to the 3' and 5' arm sequences, whereby incorporation of the primer into a double-stranded nucleic acid detectably alters the fluorescence emitted by said label moieties.
26. The process according to claim 25 wherein said interactive fluorescent label moieties comprise a fluorophore and a non-fluorescent quencher.
27. The process according to claim 20 wherein said loop sequence is 5 to 9 nucleotides in length and said complementary 3' and 5' arm sequences form a stem that is 5 to 12 nucleotides in length.
28. The process according to claim 27 wherein said priming region contains a single nucleotide subject to mutation and said loop sequence is complementary to the portion of said priming region containing said nucleotide.
29. The process according to claim 27 wherein said primer contains modified nucleotides, modified internucleotide linkages, or both.
30. The process according to claim 20 wherein said at least one primer according to claim 1 includes a terminator nucleotide between the loop and the 5' arm.
31. The process according to claim 20 wherein said priming region contains a single nucleotide subject to mutation and said loop sequence is complementary to the portion of said priming region containing said nucleotide.
32. The process according to claim 31 wherein said primer additionally comprises interactive fluorescent label moieties attached to the 3' and 5' arm sequences, whereby incorporation of the primer into a double-stranded nucleic acid detectably alters the fluorescence emitted by said label moieties.
33. The process according to claim 32 wherein said interactive fluorescent label moieties comprise a fluorophore and a non-fluorescent quencher.
34. The process according to claim 20 wherein said at one oligonucleotide primer according to claim 1 comprises a pair of primers.
35. The process according to claim 34 wherein at least one of said primers according to claim 1 has a loop sequence 5 to 9 nucleotides in length and said complementary 3' and 5' arm sequences form a stem that is 5 to 12 nucleotides in length.
36. A kit of reagents for performing amplification of a target nucleic acid sequence comprising amplification buffer, dNTPs, at least one primer according to claim 1, and instructions for performing said amplification.
37. The kit of reagents according to claim 36 wherein said at least one primer according to claim 1 includes a first primer and a second primer differing from said first primer by a single nucleotide of said loop sequence.
38. The kit of reagents according to claim 37, wherein in each of said first and second primers the loop sequence is 5 to 9 nucleotides in length and said complementary 3' and 5' arm sequences form a stem that is 5 to 12 nucleotides in length.
39. The kit of reagents according to claim 37 wherein said each of said first and second primers includes interactive fluorescent label moieties attached to the 3' end 5' arm sequences, whereby incorporation of each said primer into a double-stranded nucleic acid detectably alters the fluorescence emitted by said label moieties.
40. The kit of reagents according to claim 36 wherein in said at least one primer the loop sequence is 5 to 9 nucleotides in length and said complementary 3' and 5' arm sequences form a stem that is 5 to 12 nucleotides in length.
41. The kit of reagents according to claim 36 wherein said at least one primer according to claim 1 includes a terminator nucleotide between the loop and the 5' arm.
CA2375027A 1999-05-24 2000-05-03 High specificity primers, amplification methods and kits Expired - Lifetime CA2375027C (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US09/317,350 US6277607B1 (en) 1999-05-24 1999-05-24 High specificity primers, amplification methods and kits
US09/317,350 1999-05-24
PCT/US2000/011979 WO2000071562A1 (en) 1999-05-24 2000-05-03 High specificity primers, amplification methods and kits

Publications (2)

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CA2375027A1 true CA2375027A1 (en) 2000-11-30
CA2375027C CA2375027C (en) 2010-07-27

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US (2) US6277607B1 (en)
EP (1) EP1185546B1 (en)
JP (1) JP3843215B2 (en)
AT (1) ATE394410T1 (en)
AU (1) AU769219B2 (en)
CA (1) CA2375027C (en)
DE (1) DE60038796D1 (en)
WO (1) WO2000071562A1 (en)

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