NUCLEIC ACID ENCODING REACTIONS
First Claim
1. A method of adding adaptor molecules to each end of a plurality of target nucleic acids that comprise sticky ends, the method comprising:
- (a) annealing adaptor molecules to the sticky ends of double-stranded target nucleic acid molecules to produce annealed adaptor-target nucleic acid molecules, wherein the adaptor molecules are;
(i) hairpin structures each comprising;
an adaptor nucleotide sequence, which is linked toa nucleotide linker, which is linked toa nucleotide sequence that is capable of annealing to the adaptor nucleotide sequence and is linked toa degenerate tail sequence;
or(ii) double-stranded or single-stranded molecules each comprising;
a first adaptor nucleotide sequence, which is linked toa nucleotide linker, which is linked toa second adaptor nucleotide sequence; and
a degenerate tail sequence on each strand, wherein double-stranded molecules each comprise two degenerate tail sequences as sticky end(s);
(b) filling any gaps in the resulting annealed adaptor-target nucleic acid molecules; and
(c) ligating any adjacent nucleotide sequences in the annealed adaptor-target nucleic acid molecules to produce adaptor-modified target nucleic acid molecules.
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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
179 Claims
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1. A method of adding adaptor molecules to each end of a plurality of target nucleic acids that comprise sticky ends, the method comprising:
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(a) annealing adaptor molecules to the sticky ends of double-stranded target nucleic acid molecules to produce annealed adaptor-target nucleic acid molecules, wherein the adaptor molecules are; (i) hairpin structures each comprising; an adaptor nucleotide sequence, which is linked to a nucleotide linker, which is linked to a nucleotide sequence that is capable of annealing to the adaptor nucleotide sequence and is linked to a degenerate tail sequence;
or(ii) double-stranded or single-stranded molecules each comprising; a first adaptor nucleotide sequence, which is linked to a nucleotide linker, which is linked to a second adaptor nucleotide sequence; and a degenerate tail sequence on each strand, wherein double-stranded molecules each comprise two degenerate tail sequences as sticky end(s); (b) filling any gaps in the resulting annealed adaptor-target nucleic acid molecules; and (c) ligating any adjacent nucleotide sequences in the annealed adaptor-target nucleic acid molecules to produce adaptor-modified target nucleic acid molecules.
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2-24. -24. (canceled)
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25. A plurality of adaptor molecules, wherein the adaptor molecules are:
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(i) hairpin structures each comprising; an adaptor nucleotide sequence, which is linked to a nucleotide linker, which is linked to a nucleotide sequence that is capable of annealing to the adaptor nucleotide sequence and is linked to a degenerate tail sequence;
or(ii) double-stranded or single-stranded molecules each comprising; a first adaptor nucleotide sequence, which is linked to a nucleotide linker, which is linked to a second adaptor nucleotide sequence; and a degenerate tail sequence on each strand, wherein double-stranded molecules each comprise two degenerate tail sequences as sticky end(s). - View Dependent Claims (34)
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26-33. -33. (canceled)
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35. A method for tagging a plurality of target nucleic acids with nucleotide sequences, the method comprising:
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(a) preparing a first reaction mixture for each target nucleic acid, the first reaction mixture comprising a pair of inner primers and a pair of outer primers, wherein; (i) the inner primers comprise; a forward, inner primer comprising a first nucleotide tag, a first barcode nucleotide sequence, and a target-specific portion; and a reverse, inner primer comprising a target-specific portion, a first barcode nucleotide sequence, and a second nucleotide tag; and (ii) the outer primers comprise; a forward, outer primer comprising a second barcode nucleotide sequence and a first nucleotide tag-specific portion; and a reverse, outer primer comprising a second nucleotide tag-specific portion and a second barcode nucleotide sequence; wherein the outer primers are in excess of the inner primers; and (b) subjecting each first reaction mixture to a reaction to produce a plurality of tagged target nucleotide sequences, each comprising 5′
-second barcode nucleotide sequence-first nucleotide tag sequence-first barcode nucleotide sequence-target nucleotide sequence-first barcode nucleotide sequence-second nucleotide tag sequence-second barcode nucleotide sequence-3′
. - View Dependent Claims (40)
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36-39. -39. (canceled)
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41. A method for tagging a plurality of target nucleic acids with nucleotide sequences, the method comprising:
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(a) preparing a first reaction mixture for each target nucleic acid, the first reaction mixture comprising a pair of inner primers, a pair of stuffer primers, and a pair of outer primers, wherein; (i) the inner primers comprise; a forward, inner primer comprising a first nucleotide tag and a target-specific portion; and a reverse, inner primer comprising a target-specific portion and a second nucleotide tag; (ii) the stuffer primers comprise; a forward, stuffer primer comprising a third nucleotide tag, a first barcode nucleotide sequence, and a first nucleotide tag-specific portion; and a reverse, stuffer primer comprising a second nucleotide tag-specific portion, a first barcode nucleotide sequence, a fourth nucleotide tag; and (iii) the outer primers comprise; a forward, outer primer comprising a second barcode nucleotide sequence and a third nucleotide tag-specific portion; and a reverse, outer primer comprising a fourth nucleotide tag-specific portion and a second barcode nucleotide sequence; wherein the outer primers are in excess of the stuffer primers, which are in excess of the inner primers; and (b) subjecting each first reaction mixture to a reaction to produce a plurality of tagged target nucleotide sequences, each comprising 5′
-second barcode nucleotide sequence-third nucleotide tag sequence-first barcode nucleotide sequence-first nucleotide tag sequence-target nucleotide sequence-second nucleotide tag sequence-first barcode nucleotide sequence-fourth nucleotide tag sequence-second barcode nucleotide sequence-3′
. - View Dependent Claims (46)
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42-45. -45. (canceled)
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47. (canceled)
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48. A method for combinatorial tagging of a plurality of target nucleotide sequences, the method comprising:
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cutting a plurality of tagged target nucleotide sequences derived from target nucleic acids, each tagged target nucleotide sequence comprising an endonuclease site and a first barcode nucleotide sequence, wherein tagged target nucleotide sequences in the plurality comprise the same endonuclease site, but N different first barcode nucleotide sequences, wherein N is an integer greater than 1, with an endonuclease specific for the endonuclease site to produce a plurality of sticky-ended, tagged target nucleotide sequences; ligating a plurality of adaptors comprising a second barcode nucleotide sequence and complementary sticky ends to the plurality of sticky-ended, tagged target nucleotide sequences in a first reaction mixture, wherein the plurality of adaptors comprise M different second barcode nucleotide sequences, wherein M is an integer greater than 1, to produce a plurality of combinatorially tagged target nucleotide sequences, each comprising first and second barcode nucleotide sequences, wherein the plurality comprises N×
M different first and second barcode combinations.
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49-60. -60. (canceled)
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61. A plurality of adaptors comprising:
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a plurality of first adaptors, each comprising the same endonuclease site, N different barcode nucleotide sequences, wherein M is an integer greater than 1, a first primer binding site and a sticky end; a second adaptor comprising a second primer binding site and a sticky end; and a plurality of third adaptors comprising a second barcode nucleotide sequence and sticky ends complementary to those produced upon cutting the first adaptors at the endonuclease site, wherein the plurality of third adaptors comprise M different second barcode nucleotide sequences, wherein M is an integer greater than 1. - View Dependent Claims (62)
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63. A method for combinatorial tagging of a plurality of target nucleotide sequences, the method comprising:
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annealing a plurality of barcode primers to a plurality of tagged target nucleotide sequences derived from target nucleic acids, wherein; each tagged target nucleotide sequence comprises a nucleotide tag at one end and a first barcode nucleotide sequence, wherein tagged target nucleotide sequences in the plurality comprise the same nucleotide tag, but N different first barcode nucleotide sequences, wherein N is an integer greater than one; and each barcode primer comprises; a first tag-specific portion linked to; a second barcode nucleotide sequence linked to; a second tag-specific portion; wherein the barcode primers in the plurality each comprise the same first and second tag-specific portions, but M different second barcode nucleotide sequences, wherein M is an integer greater than one; and amplifying the tagged target nucleotide sequences in a first reaction mixture to produce a plurality of combinatorially tagged target nucleotide sequences, each comprising first and second barcode nucleotide sequences, wherein the plurality comprises N×
M different first and second barcode combinations. - View Dependent Claims (81)
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64-80. -80. (canceled)
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82-83. -83. (canceled)
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84. An assay method for detecting a plurality of target nucleic acids, the method comprising:
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preparing M first reaction mixtures that will be pooled prior to assay, wherein M is an integer greater than 1, and each first reaction mixture comprises; sample nucleic acid(s); a first, forward primer comprising a target-specific portion; a first, reverse primer comprising a target-specific portion, wherein the first, forward primer or the first, reverse primer additionally comprises a barcode nucleotide sequence, wherein each barcode nucleotide sequence in each of said M reaction mixtures is different; subjecting each first reaction mixture to a first reaction to produce a plurality of barcoded target nucleotide sequences, each comprising a target nucleotide sequence linked to a barcode nucleotide sequence. for each of the M first reaction mixtures, pooling the barcoded target nucleotide sequences to form an assay pool; subjecting the assay pool, or one or more aliquots thereof, to a second reaction using unique pairs of second primers, wherein each second primer pair comprises; a second, forward or a reverse primer that anneals to a target nucleotide sequence; and a second, reverse or a forward primer, respectively, that anneals to a barcode nucleotide sequence; and for each unique, second primer pair, determining whether a reaction product is present in the assay pool, or aliquot thereof, whereby the presence of a reaction product indicates the presence of a particular target nucleic acid in a particular first reaction mixture.
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85. An assay method for detecting a plurality of target nucleic acids, the method comprising:
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preparing M first reaction mixtures that will be pooled prior to assay, wherein M is an integer greater than 1, and each first reaction mixture comprises; sample nucleic acid(s) a first, forward primer comprising a target-specific portion; a first, reverse primer comprising a target-specific portion, wherein the first, forward primer or the first, reverse primer additionally comprises a nucleotide tag; and at least one barcode primer comprising a barcode nucleotide sequence and a nucleotide tag-specific portion, wherein the barcode primer is in excess of the first, forward and/or first, reverse primer(s), and wherein each barcode nucleotide sequence in each of said M reaction mixtures is different; subjecting each first reaction mixture to a first reaction to produce a plurality of barcoded target nucleotide sequences, each comprising a target nucleotide sequence linked to a nucleotide tag, which is linked to a barcode nucleotide sequence. for each of the M reaction mixtures, pooling the barcoded target nucleotide sequences to form an assay pool; subjecting the assay pool, or one or more aliquots thereof, to a second reaction using unique pairs of second primers, wherein each second primer pair comprises; a second, forward or a reverse primer that anneals to a target nucleotide sequence; and a second, reverse or a forward primer, respectively, that anneals to a barcode nucleotide sequence; and for each unique, second primer pair, determining whether a reaction product is present in the assay pool, or aliquot thereof, whereby the presence of a reaction product indicates the presence of a particular target nucleic acid in a particular first reaction mixture.
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86-131. -131. (canceled)
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132. 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′
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133-167. -167. (canceled)
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168. A kit for use in amplifying a target nucleic acid, the kit comprising:
a first set of outer primers, wherein the set comprises; a first outer, forward primer comprising a portion specific for a first primer binding site; and a first outer, reverse primer comprising a barcode nucleotide sequence and a portion specific for a second primer binding site, wherein the first and second primer binding sites are different; 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. - View Dependent Claims (169, 170, 171, 172, 173, 174, 175, 176, 177)
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178. A method for detecting, and/or quantifying the relative amounts of, at least two different target nucleic acids in a nucleic acid sample, the method comprising:
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producing first and second tagged target nucleotide sequences from first and second target nucleic acids in the sample, the first tagged target nucleotide sequence comprising a first nucleotide tag; and the second tagged target nucleotide sequence comprising a second nucleotide tag, wherein the first and second nucleotide tags are different; subjecting the tagged target nucleotide sequences to; a first primer extension reaction using a first primer that anneals to the first nucleotide tag; and a second primer extension reaction using a second primer that anneals to the second nucleotide tag; and detecting and/or quantifying; a signal that indicates extension of the first primer, and a signal that indicates extension of the second primer wherein the a signal for a given primer indicates the presence, and/or relative amount of, the corresponding target nucleic acid.
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179-209. -209. (canceled)
Specification