Nucleic acid encoding reactions

Patent metadata

Field	Value
Publication number	US-2016208322-A1
Application number	US-201514723872-A
Country	US
Kind code	A1
Filing date	May 28, 2015
Priority date	May 20, 2011
Publication date	Jul 21, 2016
Grant date	—

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.

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 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 . (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).

26 - 33 . (canceled)

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.

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′.

36 - 39 . (canceled)

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.

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′.

42 - 45 . (canceled)

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.

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

Assignees

• Fluidigm Corp

Inventors

• Anderson Megan
• Chen Peilin
• Fowler Brian
• Jones Robert C
• Kaper Fiona
• Lebofsky Ronald
• May Andrew

Classifications

• C12N15/65 Primary

  using markers (enzymes used as markers C12N15/52) · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6855 Primary

  Ligating adaptors · CPC title

• C12N15/10

  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 title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title