Specific inhibition of the polymerase chain reaction using a non-extendable oligonucleotide blocker

• US 5,849,497 A
• Filed: 04/03/1997
• Issued: 12/15/1998
• Est. Priority Date: 04/03/1997
• Status: Expired due to Fees

• Current Assignee: The Research Foundation for The State University of New York (State University of New York)
• Original Assignee: The Research Foundation for The State University of New York (State University of New York)
• Inventors: Steinman, Charles
• Primary Examiner(s): Elliott, George C.
• Assistant Examiner(s): LARSON, THOMAS GEORGE
• Application Number:
  US08/832,449
• Time in Patent Office: 621 Days
• Field of Search: 435/6, 435/91.3, 436/94, 536/24.32, 536/24.33, 935/17, 935/77
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm

Abstract

A process is disclosed for inhibiting the amplification of a DNA template by subjecting a sample of biological material containing nucleic acid to the polymerase chain reaction (PCR) using a DNA polymerase deficient in 5'"'"' exonuclease activity. The method comprises forming a PCR admixture comprising the DNA template, first and second oligonucleotide primers which are complementary to separated regions of the nucleic acid template, a non-extendable oligonucleotide blocker which is complementary to the inter-primer region of the DNA, and the DNA polymerase lacking 5'"'"' exonuclease activity, and subjecting the PCR admixture to at least one PCR thermocycle. The DNA polymerase lacking 5'"'"' exonuclease activity is incapable of excising the non-extendable blocker which anneals to the DNA template during the PCR, thereby inhibiting amplification which would otherwise occur during the PCR. Preferably, the DNA polymerase lacking 5'"'"' exonuclease activity is the Stoffel fragment of Taq polymerase. The method may be adapted for detecting whether DNA from specific pathogens is present in a sample material.

Claims

1. A method of specific inhibition of amplification of at least one target sequence located on at least one DNA sequence in the polymerase chain reaction (PCR), comprising:

forming a PCR admixture comprising at least one DNA target sequence, at least first and second oligonucleotide primers which are complementary to first and second separated regions on said target sequence, at least one non-extendable oligonucleotide blocker complementary to at least a portion of an inter-primer region located between said first primer region and the sequence complementary to said second primer region on said target sequence, said oligonucleotide blocker comprising a plurality of nucleotides connected to each other by phosphodiester linkages, appropriate buffers and nucleic acid precursors, and a nucleic acid polymerase which lacks 5'"'"' exonuclease activity; and

subjecting said PCR admixture to at least one PCR thermocycle comprising hybridization, primer extension and denaturation, whereby amplification of said target sequence is inhibited when said at least one non-extendable oligonucleotide blocker anneals to said at least one inter-primer regions of said target sequence.

2. The method of claim 1 wherein said non-extendable oligonucleotide blocker has at least one 3'"'"' mismatch relative to said target sequence.

3. The method of claim 1 wherein said nucleic acid polymerase is the Stoffel fragment of Taq polymerase.

4. The method of claim 1 wherein said non-extendable oligonucleotide blocker has a higher melting point than the melting point of said first primer.

5. The method of claim 4 wherein said PCR thermocycle includes an incubation step at a temperature higher than the annealing point of said first primer and lower than the annealing point of said non-extendable oligonucleotide blocker.

6. The method of claim 5 wherein said PCR admixture includes a molar excess of said non-extendable oligonucleotide blocker over said first and second primers.

7. The method of claim 1 further comprising detecting amplification of said target sequence.

8. The method of claim 1 wherein said non-extendable oligonucleotide blocker terminates at its 5'"'"' end in a 2'"'"',3'"'"'-dideoxynucleotide.

9. A method of detecting the presence of at least one specific DNA target sequence located on at least one DNA sequence in the polymerase chain reaction (PCR), comprising:

forming a PCR admixture comprising at least first and second oligonucleotide primers which are complementary to first and second separated regions on said target sequence, at least one non-extendable oligonucleotide blocker complementary to at least a portion of at least one inter-primer region located between said first primer region and a sequence complementary to said second oligonucleotide primer on said target sequence, said oligonucleotide blocker comprising a plurality of nucleotides connected to each other by phosphodiester linkages, appropriate buffers and nucleic acid precursors, and a DNA polymerase which lacks 5'"'"' exonuclease activity;

adding an unknown sample to be tested for the presence of said target to said PCR admixture;

subjecting said PCR admixture including said unknown sample to at least one PCR thermocycle comprising hybridization, primer extension and denaturation, wherein amplification will be inhibited during said PCR thermocycle when said non-extendable oligonucleotide blocker anneals to said at least one inter-primer of said target sequence; and

detecting whether amplification of said target sequence has been inhibited by comparing said PCR admixture including said unknown sample with a parallel reaction in which said non-extendable oligonucleotide blocker is omitted, wherein the absence of amplification of said target sequence in said PCR admixture containing said unknown sample together with amplification of said target in said parallel reaction indicates the presence of said target sequence in said PCR admixture comprising said unknown sample.

10. The method of claim 9 wherein said DNA polymerase is the Stoffel fragment of Taq polymerase.

11. The method of claim 9 wherein said PCR thermocycle includes an incubation step at a temperature higher than the annealing point of said first primer and lower than the annealing point of said non-extendable oligonucleotide blocker.

12. The method of claim 9 wherein said first and second primers are complementary to first and second primer regions present on nucleic acid of E. coli, S. aureus, or N. gonorrhoea;

and

said non-extendable oligonucleotide blocker is complementary to at least a portion of the inter-primer region present on the nucleic acid of E. coli, S. aureus, or N. gonorrhoea.

13. The method of claim 9 wherein said PCR thermocycle includes a hybridization step at a temperature in the range of 50°

to 80°

C., a primer extension step at a temperature in the range of about 50°

-80°

C., and a denaturation step at a temperature in the range of about 90°

to 100°

C.

14. The method of claim 9 wherein said PCR thermocycle is begun at a temperature of about 85°

to 100°

C.

15. The method of claim 9 wherein said PCR admixture includes a molar excess of said non-extendable oligonucleotide blocker relative to said first primer.

16. The method of claim 9 wherein said non-extendable oligonucleotide blocker has at least one 3'"'"' mismatch relative to said target sequence.

17. The method of claim 9 wherein said non-extendable oligonucleotide blocker terminates at its 5'"'"' end in a 2'"'"',3'"'"'-dideoxynucleotide.

18. A kit for detecting the presence of a specific nucleic acid sequence in a sample, comprising:

a PCR admixture comprising at least first and second oligonucleotide primers which are complementary to first and second separated regions on said specific nucleic acid sequence, at least one non-extendable oligonucleotide blocker complementary to at least a portion of an inter-primer region between said first primer region and said region complementary to said second primer region on said specific nucleic acid sequence, said oligonucleotide blocker comprising a plurality of nucleotides connected to each other by phosphodiester linkages, and a polymerase lacking 5'"'"' exonuclease activity.

19. The kit of claim 18 further comprising electrophoresis reagents.

20. The kit of claim 18 further comprising a second pair of primers, primer 3 and primer 4, targeting a region different from that targeted by primers 1 and 2 and that includes an inter-primer region to which said non-extendable oligonucleotide blocker does not bind and whose amplification would therefore not be inhibited by said non-extendable oligonucleotide blocker, in order to control for nonspecific inhibition of amplification.

21. The kit of claim 18 wherein said first and second primers that are complementary to separated regions on nucleic acid of E. coli, S. aureus, or N. gonorrhoea;

and

non-extendable oligonucleotide blockers that are complementary to at least a portion of the inter-primer regions of nucleic acid of E. coli, S. aureus, and N. gonorrhoea.

22. The kit of claim 18 wherein said polymerase is the Stoffel fragment of Taq polymerase.