Protection of barcodes during DNA amplification using molecular hairpins
First Claim
Patent Images
1. A method of amplifying a target nucleic acid in a sample comprising:
- a. contacting a sample comprising a target nucleic acid with a target-specific hairpin barcode forward primer and a target-specific reverse primer,wherein the hairpin barcode forward primer comprises, in a 5′
to 3′
direction;
a 5′
stem sequence, an adaptor sequence, a barcode sequence, a 3′
stem sequence, and a 3′
target-specific sequence, wherein the 5′
stem sequence and the 3′
stem sequence each comprise sequence complementary to each other and the complementary sequences hybridize to each other under a closed annealing temperature and do not hybridize to each other at an open annealing temperature;
b. amplifying the target nucleic acid by performing 2-5 cycles of PCR pre-amplification on the target nucleic acid, wherein the 2-5 cycles of PCR pre-amplification have an annealing temperature less than or equal to the closed annealing temperature of the hairpin barcode forward primer, to generate a plurality of pre-amplification target nucleic acids;
c. contacting the plurality of pre-amplification target nucleic acids with an adaptor-specific forward primer and an adaptor-specific reverse primer; and
d. amplifying the pre-amplification target nucleic acid by performing at least 10 cycles of PCR amplification on the pre-amplification target nucleic acids, wherein at least 3 of the at least 10 cycles of PCR-based amplification have an annealing temperature greater than or equal to the open annealing temperature of the hairpin barcode forward primer, to generate a plurality of target nucleic acid amplicons, wherein the target nucleic acid amplicons comprise the adaptor sequence and the barcode sequence.
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Abstract
Described herein are approaches for the improved detection, identification, and/or quantification of target nucleic acids. These approaches provide a means of detecting, identifying, and/or quantifying rare target nucleic acid molecules, including DNA and RNA molecules, from the same sample, and in the same reaction, by using “hairpin barcode primers,” as the term is defined herein, to incorporate unique barcodes into target nucleic acids in a PCR pre-amplification step.
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Citations
21 Claims
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1. A method of amplifying a target nucleic acid in a sample comprising:
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a. contacting a sample comprising a target nucleic acid with a target-specific hairpin barcode forward primer and a target-specific reverse primer, wherein the hairpin barcode forward primer comprises, in a 5′
to 3′
direction;
a 5′
stem sequence, an adaptor sequence, a barcode sequence, a 3′
stem sequence, and a 3′
target-specific sequence, wherein the 5′
stem sequence and the 3′
stem sequence each comprise sequence complementary to each other and the complementary sequences hybridize to each other under a closed annealing temperature and do not hybridize to each other at an open annealing temperature;b. amplifying the target nucleic acid by performing 2-5 cycles of PCR pre-amplification on the target nucleic acid, wherein the 2-5 cycles of PCR pre-amplification have an annealing temperature less than or equal to the closed annealing temperature of the hairpin barcode forward primer, to generate a plurality of pre-amplification target nucleic acids; c. contacting the plurality of pre-amplification target nucleic acids with an adaptor-specific forward primer and an adaptor-specific reverse primer; and d. amplifying the pre-amplification target nucleic acid by performing at least 10 cycles of PCR amplification on the pre-amplification target nucleic acids, wherein at least 3 of the at least 10 cycles of PCR-based amplification have an annealing temperature greater than or equal to the open annealing temperature of the hairpin barcode forward primer, to generate a plurality of target nucleic acid amplicons, wherein the target nucleic acid amplicons comprise the adaptor sequence and the barcode sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method of pre-amplifying a target nucleic acid in a sample prior to amplification comprising:
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a. contacting a sample comprising a target nucleic acid with a target-specific hairpin barcode forward primer and a target-specific reverse primer, wherein the hairpin barcode forward primer comprises, in a 5′
to 3′
direction;
a 5′
stem sequence, a sequence to be protected, a 3′
stem sequence, and a 3′
target-specific sequence, wherein the 5′
stem sequence and the 3′
stem sequence each comprise sequence complementary to each other, and the complementary sequences hybridize to each other under a closed annealing temperature and do not hybridize to each other at an open annealing temperature, andwherein the sequence to be protected comprises, in the 5′
to 3′
direction, a barcode sequence and an adaptor sequence; andb. pre-amplifying the target nucleic acid by performing at least one cycle of PCR pre-amplification on the target nucleic acid, wherein the at least one cycle of PCR pre-amplification has an annealing temperature less than or equal to the closed annealing temperature of the hairpin barcode forward primer, thereby generating a plurality of pre-amplification target nucleic acids. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20)
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12. A method of amplifying a target nucleic acid in a sample comprising:
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a. contacting a sample comprising a target nucleic acid with a target-specific hairpin barcode forward primer and a target-specific reverse primer, wherein the hairpin barcode forward primer comprises, in a 5′
to 3′
direction;
a 5′
stem sequence, a sequence to be protected, a 3′
stem sequence, and a 3′
target-specific sequence, wherein the 5′
stem sequence and the 3′
stem sequence each comprise sequence complementary to each other, and the complementary sequences hybridize to each other under a closed annealing temperature and do not hybridize to each other at an open annealing temperature, andwherein the sequence to be protected comprises, in the 5′
to 3′
direction, a barcode sequence and an adaptor sequence;b. pre-amplifying the target nucleic acid by performing at least one cycle of PCR pre-amplification on the target nucleic acid, wherein the at least one cycle of PCR pre-amplification has an annealing temperature less than or equal to the closed annealing temperature of the hairpin barcode forward primer, to generate a plurality of pre-amplification target nucleic acids; c. contacting the plurality of pre-amplification target nucleic acids with an adaptor-specific forward primer and an adaptor-specific reverse primer; and d. amplifying the pre-amplification target nucleic acid by performing at least 10 cycles of PCR amplification on the pre-amplification target nucleic acids, wherein the at least 10 cycles of PCR-based amplification have an annealing temperature greater than or equal to the open annealing temperature of the hairpin barcode forward primer, to generate a plurality of target nucleic acid amplicons, wherein the target nucleic acid amplicons comprise the adaptor sequence and the barcode sequence.
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21. A method of producing a pre-amplified target nucleic acid comprising:
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a. contacting a sample comprising a target nucleic acid with a target-specific hairpin barcode forward primer and a target-specific reverse primer, wherein the hairpin barcode forward primer comprises, in a 5′
to 3′
direction;
a 5′
stem sequence, a sequence to be protected, a 3′
stem sequence, and a 3′
target-specific sequence, wherein the 5′
stem sequence and the 3′
stem sequence each comprise sequence complementary to each other, and the complementary sequences hybridize to each other under a closed annealing temperature and do not hybridize to each other at an open annealing temperature, andwherein the sequence to be protected comprises, in the 5′
to 3′
direction, a barcode sequence and an adaptor sequence; andb. pre-amplifying the target nucleic acid by performing at least one cycle of PCR pre-amplification on the target nucleic acid, wherein the at least one cycle of PCR pre-amplification has an annealing temperature less than or equal to the closed annealing temperature of the hairpin barcode forward primer, thereby generating a plurality of pre-amplification target nucleic acids.
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Specification