Quantitative PCR using blocking oligonucleotides
First Claim
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1. A method for determining the number of copies of a target nucleic acid relative to the number of copies of a reference nucleic acid in a biological specimen, which method comprises the steps of:
- (a) amplifying the target nucleic acid in said specimen with a polymerase, a first primer specific for said target nucleic acid with or without a segment noncontiguous to a first priming sequence, a second primer specific for said target nucleic acid with or without a segment noncontiguous to a second priming sequence in the presence of a first oligonucleotide which is incapable of acting as a primer for said polymerase, wherein said first oligonucleotide has at least 5 consecutive nucleotides fully complementary to at least 5 consecutive nucleotides of said first primer;
(b) measuring the amplification of the target nucleic acid;
(c) amplifying the reference nucleic acid in said specimen with a polymerase, a third primer specific for said reference nucleic acid with or without a segment noncontiguous to a third priming sequence, and a fourth primer specific for said reference nucleic acid sequence with or without a segment noncontiguous to a fourth priming sequence in the presence of a second oligonucleotide which is incapable of acting as a primer for said polymerase, wherein said second oligonucleotide has at least 5 consecutive nucleotides fully complementary to at least 5 consecutive nucleotides of said third primer;
(d) measuring the amplification of the reference nucleic acid; and
(e) comparing the amplification of said target nucleic acid sequence to the amplification of said reference nucleic acid sequence in said specimen to determine the number of copies of the target nucleic acid relative to the number of copies of the reference nucleic acid.
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Abstract
Disclosed is a method for determining the number of copies of a target nucleic acid relative to the number of copies of a reference nucleic acid in a biological specimen. The method includes amplifying the target nucleic acid, measuring the amplification of the target nucleic acid, amplifying the reference nucleic acid, measuring the amplification of the target nucleic acid, and comparing the amplification of the target nucleic acid sequence to the amplification of the reference nucleic acid sequence.
73 Citations
23 Claims
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1. A method for determining the number of copies of a target nucleic acid relative to the number of copies of a reference nucleic acid in a biological specimen, which method comprises the steps of:
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(a) amplifying the target nucleic acid in said specimen with a polymerase, a first primer specific for said target nucleic acid with or without a segment noncontiguous to a first priming sequence, a second primer specific for said target nucleic acid with or without a segment noncontiguous to a second priming sequence in the presence of a first oligonucleotide which is incapable of acting as a primer for said polymerase, wherein said first oligonucleotide has at least 5 consecutive nucleotides fully complementary to at least 5 consecutive nucleotides of said first primer; (b) measuring the amplification of the target nucleic acid; (c) amplifying the reference nucleic acid in said specimen with a polymerase, a third primer specific for said reference nucleic acid with or without a segment noncontiguous to a third priming sequence, and a fourth primer specific for said reference nucleic acid sequence with or without a segment noncontiguous to a fourth priming sequence in the presence of a second oligonucleotide which is incapable of acting as a primer for said polymerase, wherein said second oligonucleotide has at least 5 consecutive nucleotides fully complementary to at least 5 consecutive nucleotides of said third primer; (d) measuring the amplification of the reference nucleic acid; and (e) comparing the amplification of said target nucleic acid sequence to the amplification of said reference nucleic acid sequence in said specimen to determine the number of copies of the target nucleic acid relative to the number of copies of the reference nucleic acid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A kit for detecting the copy number of a target nucleic acid relative to a reference nucleic acid, said kit comprising:
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a first primer with or without a segment noncontiguous to a first priming sequence, a second primer with or without a segment noncontiguous to a second priming sequence, which said first and second primers are to be used with a polymerase for the amplification of the target nucleic acid; a third primer with or without a segment noncontiguous to a third priming sequence, a fourth primer with or without a segment noncontiguous to a fourth priming sequence, which said third and fourth primers are to be used with a polymerase for the amplification of the reference nucleic acid; a first oligonucleotide which is incapable of acting as a primer for said polymerase and has at least 5 consecutive nucleotides fully complementary to at least 5 consecutive nucleotides of said first primer; and a second oligonucleotide which is incapable of acting as a primer for said polymerase and has at least 5 consecutive nucleotides fully complementary to at least 5 consecutive nucleotides of said third primer, wherein each of said first primer, said second primer, said third primer, said fourth primer, said first oligonucleotide, and said second oligonucleotide contains 10-50 nucleotides. - View Dependent Claims (22, 23)
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Specification