NUCLEIC ACID ENCODING REACTIONS

• US 20130005585A1
• Filed: 05/21/2012
• Published: 01/03/2013
• Est. Priority Date: 05/20/2011
• Status: Active Grant

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Anderson, Megan, Chen, Peilin, Fowler, Brian, Jones, Robert C., May, Andrew, Kaper, Fiona, Lebofsky, Ronald
• Granted Patent: US 9,074,204 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/65 using markers enzymes used as markers C12N15/52
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation

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.

Claims

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 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.

2-24. -24. (canceled)

25. A plurality of adaptor 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).

34. A kit comprising the plurality of adaptor molecules of claim 25 and one or more components selected from the group consisting of a DNAse enzyme, an exonuclease, an endonuclease, a polymerase, and a ligase.

26-33. -33. (canceled)

35. A method for tagging a plurality of target nucleic acids with nucleotide sequences, the method comprising:

(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′

.

40. A kit for performing the method of claim 35, wherein the kit comprises a polymerase and:

(i) inner primers comprising;

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) outer primers comprising;

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.

36-39. -39. (canceled)

41. A method for tagging a plurality of target nucleic acids with nucleotide sequences, the method comprising:

(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′

.

46. A kit for performing the method of claim 41, wherein the kit comprises a polymerase and:

(i) inner primers comprising;

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) stuffer primers comprising;

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) outer primers comprising;

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.

42-45. -45. (canceled)

47. (canceled)

48. A method for combinatorial tagging of a plurality of target nucleotide sequences, the method comprising:

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.

49-60. -60. (canceled)

61. A plurality of adaptors comprising:

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.

62. A kit comprising the adaptors of claim 61 and one or more components selected from the group consisting of:

an endonuclease specific for the endonuclease site in the first adaptors and a ligase.

63. A method for combinatorial tagging of a plurality of target nucleotide sequences, the method comprising:

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.

81. A kit for performing the method of claim 63, the kit comprising:

one or more nucleotide tags(s); and

a plurality of barcode primers, wherein each barcode primer comprises;

a first portion that is specific for a first portion of the nucleotide tag(s) linked to;

a barcode nucleotide sequence that does not anneal to the nucleotide tag(s) linked to;

a second portion that is specific for a second portion of the nucleotide tag(s);

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.

64-80. -80. (canceled)

82-83. -83. (canceled)

84. An assay method for detecting a plurality of target nucleic acids, the method comprising:

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.

85. An assay method for detecting a plurality of target nucleic acids, the method comprising:

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.

86-131. -131. (canceled)

132. 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′

.

133. The method of claim 132, wherein the barcode nucleotide sequence in each target amplicon is the same, and wherein each target amplicon comprises only one barcode nucleotide sequence.

134. The method of claim 132, wherein the first and second primer binding sites are binding sites for DNA sequencing primers.

135. The method of claim 132, wherein the outer primers each additionally comprise an additional nucleotide sequence, wherein:

the first outer, forward primer comprises a first additional nucleotide sequence, and the first outer, reverse primer comprises a second additional nucleotide sequence; and

the second outer, forward primer comprises the second additional nucleotide sequence, and the second outer, reverse primer comprises the first additional nucleotide sequence; and

the first and second additional nucleotide sequences are different; and

said amplifying produces two target amplicons, wherein;

a first target amplicon comprises 5′

-first additional nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-barcode nucleotide sequence-second additional nucleotide sequence-3′

; and

a second target amplicon comprises 5′

-second additional nucleotide sequence-barcode nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-first additional nucleotide sequence 3′

.

136. The method of claim 135, wherein the first or second additional nucleotide sequence comprises a primer binding site.

137. The method of claim 136, wherein the first and second additional nucleotide sequences comprise a primer binding site.

138. The method of claim 135, wherein the first set of outer primers comprises PE1-CS1 and PE2-BC-CS2, and the second set of outer primers comprises PE1-CS2 and PE2-BC-CS1 (Table, Example 9).

139. The method of claim 132, wherein said amplifying is carried out in a single amplification reaction.

140. The method of claim 132, wherein said amplifying comprises employing the inner primers in a first amplification reaction and employing the outer primers in a second amplification reaction, wherein said second amplification reaction is separate from the first.

141. The method of claim 140, wherein the second amplification reaction comprises two separate amplification reactions, wherein one employs the first set of outer primers and the other employs the second set of outer primers.

142. The method of claim 141, wherein the target amplicons produced in the two separate second amplification reactions are pooled.

143. The method of claim 132, wherein the method comprises amplifying a plurality of target nucleic acids.

144. The method of claim 143, wherein the plurality of target nucleic acids is selected from the group consisting of genomic DNA, cDNA, fragmented DNA, DNA reverse-transcribed from RNA, a DNA library, and nucleic acids is extracted or amplified from a cell, a bodily fluid or a tissue sample.

145. The method of claim 144, wherein the plurality of target nucleic acids is amplified from a formalin-fixed, paraffin-embedded tissue sample.

146. The method of claim 132, wherein the method additionally comprises sequencing the target amplicons.

147. The method of claim 143, wherein the method comprises amplifying the target amplicons using primers that bind to the first and second additional nucleotide sequences to produce templates for DNA sequencing.

148. The method of claim 147, wherein one or both primers that bind to the first and second additional nucleotide sequences are immobilized on a substrate.

149. The method of claim 143, wherein the amplification is carried out by isothermal nucleic acid amplification.

150. The method of claim 147, wherein the method comprises performing DNA sequencing using the templates and primers that bind to the first and second primer binding sites and prime sequencing of the target nucleotide sequence(s).

151. The method of claim 150, wherein the primers that bind to the first and second primer binding sites and prime sequencing of the target nucleotide sequence(s) are present in substantially equal amounts.

152. The method of claim 147, wherein the method comprises performing DNA sequencing using the templates and primers that bind to the first and second primer binding sites and prime sequencing of the barcode nucleotide sequences(s).

153. The method of claim 152, wherein the primers that bind to the first and second primer binding sites and prime sequencing of the barcode nucleotide sequences(s) are present in substantially equal amounts.

154. The method of claim 150, wherein the method comprises performing DNA sequencing using the templates and primers that bind to the first and second primer binding sites and prime sequencing of the barcode nucleotide sequences(s), wherein said primers are reverse complements of the primers that prime sequencing of the target nucleotide sequences.

155. The method of claim 154, wherein the primers employed to prime sequencing of the target nucleotide sequence(s) and barcode nucleotide sequence(s) comprise CS1, CS2, CS1rc, and CS2rc (Table 4, Example 9).

156. The method of claim 132, wherein the barcode nucleotide sequence is selected so as to avoid substantial annealing to the target nucleic acids.

157. The method of claim 132, wherein the barcode nucleotide sequence identifies a particular sample.

158. The method of claim 147, wherein at least 50 percent of the sequences determined from DNA sequencing are present at greater than 50 percent of the average number of copies of sequences and less than 2-fold the average number of copies of sequences.

159. The method of claim 147, wherein at least 70 percent of the sequences determined from DNA sequencing are present at greater than 50 percent of the average number of copies of sequences and less than 2-fold the average number of copies of sequences.

160. The method of claim 147, wherein at least 90 percent of the sequences determined from DNA sequencing are present at greater than 50 percent of the average number of copies of sequences and less than 2-fold the average number of copies of sequences.

161. The method of claim 132, wherein the average length of the target amplicons is less than 200 bases.

162. The method of claim 132, wherein the first amplification reaction is carried out in a volume in the range of about 1 picoliter to about 50 nanoliters.

163. The method of claim 132, wherein the first amplification reaction is carried out in a volume in the range of about 5 picoliters to about 25 nanoliters.

164. The method of claim 132, wherein the first amplification reaction(s) is/are formed in, or distributed into, separate compartments of a microfluidic device prior to amplification.

165. The method of claim 164, wherein the microfluidic device is fabricated, at least in part, from an elastomeric material.

166. The method of claim 132, wherein the first amplification reaction(s) is/are carried out in (a) fluid droplet(s).

167. The method of claim 166, wherein a plurality of first amplification reactions are carried out in fluid droplets in an emulsion.

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.

169. The kit of claim 168, wherein the first and second primer binding sites are binding sites for DNA sequencing primers.

170. The kit of claim 168, wherein the outer primers each additionally comprise an additional nucleotide sequence, wherein:

the first outer, forward primer comprises a first additional nucleotide sequence, and the first outer, reverse primer comprises a second additional nucleotide sequence; and

the second outer, forward primer comprises the second additional nucleotide sequence, and the second outer, reverse primer comprises the first additional nucleotide sequence; and

the first and second additional nucleotide sequences are different.

171. The kit of claim 170, wherein the first set of outer primers comprises PE1-CS1 and PE2-BC-CS2, and the second set of outer primers comprises PE1-CS2 and PE2-BC-CS1 (Table, Example 9).

172. The kit of claim 168, additionally comprising:

a set of inner primers, wherein the set comprises;

an inner, forward primer comprising a target-specific portion and the first primer binding site; and

an inner, reverse primer comprising a target-specific portion and the second primer binding site.

173. The kit of claim 172, comprising a plurality of sets of inner primers, each specific for a different target nucleic acid.

174. The kit of claim 168, additionally comprising DNA sequencing primers that bind to the first and second primer binding sites and prime sequencing of the target nucleotide sequence(s).

175. The kit of claim 168, additionally comprising DNA sequencing primers that bind to the first and second primer binding sites and prime sequencing of the barcode nucleotide sequence(s).

176. The kit of claim 175, wherein the primers that bind to the first and second primer binding sites and prime sequencing of the barcode nucleotide sequences(s) are reverse complements of the primers that prime sequencing of the target nucleotide sequences.

177. The kit of claim 176, wherein the primers employed to prime sequencing of the target nucleotide sequence(s) and barcode nucleotide sequence(s) comprise CS1, CS2, CS1rc, and CS2rc (Table 4, Example 9).

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:

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.

179-209. -209. (canceled)