Nucleic acid encoding reactions
First Claim
1. A method for amplifying a target nucleic acid, the method comprising:
- amplifying a target nucleic acid using;
a set of inner primers, wherein the set comprises;
an inner, forward primer comprising a target-specific portion and a first primer binding site;
an inner, reverse primer comprising a target-specific portion and a second primer binding site, wherein the first and second primer binding sites are different;
a first set of outer primers, wherein the set comprises;
a first outer, forward primer comprising a portion specific for the first primer binding site; and
a first outer, reverse primer comprising a barcode nucleotide sequence and a portion specific for the second primer binding site;
a second set of outer primers, wherein the set comprises;
a second outer, forward primer comprising a barcode nucleotide sequence and a portion specific for the first primer binding site; and
a second outer, reverse primer comprising a portion specific for the second primer binding site;
to produce two target amplicons, wherein;
a first target amplicon comprises 5′
-first primer binding site-target nucleotide sequence-second primer binding site-barcode nucleotide sequence-3′
; and
a second target amplicon comprises 5′
-barcode nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-3′
;
wherein the method comprises amplifying a plurality of target nucleic acids and produces target amplicons having common first and common second primer binding sites, wherein at least some target amplicons have different barcode nucleotide sequences.
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Abstract
Described herein are methods useful for incorporating one or more adaptors and/or nucleotide tag(s) and/or barcode nucleotide sequence(s) one, or typically more, target nucleotide sequences. In particular embodiments, nucleic acid fragments having adaptors, e.g., suitable for use in high-throughput DNA sequencing are generated. In other embodiments, information about a reaction mixture is encoded into a reaction product. Also described herein are methods and kits useful for amplifying one or more target nucleic acids in preparation for applications such as bidirectional nucleic acid sequencing. In particular embodiments, methods of the invention entail additionally carrying out bidirectional DNA sequencing. Also described herein are methods for encoding and detecting and/or quantifying alleles by primer extension.
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Citations
35 Claims
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1. A method for amplifying a target nucleic acid, the method comprising:
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amplifying a target nucleic acid using; a set of inner primers, wherein the set comprises; an inner, forward primer comprising a target-specific portion and a first primer binding site; an inner, reverse primer comprising a target-specific portion and a second primer binding site, wherein the first and second primer binding sites are different; a first set of outer primers, wherein the set comprises; a first outer, forward primer comprising a portion specific for the first primer binding site; and a first outer, reverse primer comprising a barcode nucleotide sequence and a portion specific for the second primer binding site; a second set of outer primers, wherein the set comprises; a second outer, forward primer comprising a barcode nucleotide sequence and a portion specific for the first primer binding site; and a second outer, reverse primer comprising a portion specific for the second primer binding site; to produce two target amplicons, wherein; a first target amplicon comprises 5′
-first primer binding site-target nucleotide sequence-second primer binding site-barcode nucleotide sequence-3′
; anda second target amplicon comprises 5′
-barcode nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-3′
;wherein the method comprises amplifying a plurality of target nucleic acids and produces target amplicons having common first and common second primer binding sites, wherein at least some target amplicons have different barcode nucleotide sequences. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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Specification