Specific inhibition of the polymerase chain reaction using a non-extendable oligonucleotide blocker
First Claim
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 Assignments
0 Petitions
Accused Products
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.
-
Citations
22 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
-
-
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. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16, 17)
-
-
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. - View Dependent Claims (19, 20, 21, 22)
Specification