Methods for sequencing nucleic acids

Patent metadata

Field	Value
Publication number	US-2016369334-A1
Application number	US-201415100919-A
Country	US
Kind code	A1
Filing date	Dec 5, 2014
Priority date	Dec 5, 2013
Publication date	Dec 22, 2016
Grant date	—

Abstract

Provided herein are methods and compositions for the sequencing of long nucleic acids, such as DNA. The methods and compositions are suited for the spatial labeling and sequencing of long nucleic acid molecules.

First claim

1 . A method for making a modified surface, comprising:
(a) providing a surface; (b) covalently bonding initiator species to said surface; (c) conducting surface initiated polymerization of a polymer from said initiator species, thereby producing a polymer coating comprising a plurality of polymer chains; and (d) coupling markers to said polymer coating.

2 . The method of claim 1 , wherein said surface is selected from the group consisting of glass, silica, titanium oxide, aluminum oxide, indium tin oxide (ITO), silicon, polydimethylsiloxane (PDMS), polystyrene, polycyclicolefins, polymethylmethacrylate (PMMA), titanium, and gold.

3 - 4 . (canceled)

5 . The method of claim 1 , wherein said surface is selected from the group consisting of flow cells, sequencing flow cells, flow channels, micro fluidic channels, capillary tubes, piezoelectric surfaces, wells, microwells, microwell arrays, microarrays, chips, wafers, nonmagnetic beads, magnetic beads, ferromagnetic beads, paramagnetic beads, superparamagnetic beads, and polymer gels.

6 . The method of claim 1 , wherein said initiator species comprises an organosilane.

7 . The method of claim 1 , wherein said initiator species comprises the molecule shown in FIG. 40 .

8 . The method of claim 1 , wherein said polymer species is polyacrylamide, PMMA, or polystyrene.

9 - 10 . (canceled)

11 . The method of claim 1 , wherein said conducting surface initiated polymerization is atom-transfer radical polymerization (ATRP) or reversible addition fragmentation chain-transfer (RAFT).

12 . (canceled)

13 . The method of claim 1 , wherein said markers are oligonucleotides or 5′ acrydite modified oligonucleotides.

14 . (canceled)

15 . A composition for transferring an array, comprising:
(a) a substrate; (b) a coating coupled to said substrate; and (c) a plurality of first recipient oligonucleotides coupled to said coating, wherein each of said plurality of first recipient oligonucleotides comprises sequence complementary to a first adaptor sequence appended to each of a plurality of template oligonucleotides, wherein the plurality of template oligonucleotides is present on an array to be transferred.

16 . The composition of claim 15 , further comprising:
(d) a plurality of second recipient oligonucleotides coupled to said coating, wherein each of said plurality of second recipient oligonucleotides comprises sequence complementary to a second adaptor sequence of template oligonucleotides to be transferred.

17 . The composition of claim 15 , wherein said first adaptor sequence is located at or near the 3′ end of said template oligonucleotides to be transferred.

18 . The composition of claim 15 , wherein said first adaptor sequence is located at or near the 5′ end of said template oligonucleotides to be transferred.

19 . The composition of claim 16 , wherein said second adaptor sequence is located at or near the 3′ end of said template oligonucleotides to be transferred.

20 . The composition of claim 16 , wherein said second adaptor sequence is located at or near the 5′ end of said template oligonucleotides to be transferred.

21 . The composition of claim 15 , wherein said coating comprises a polymer gel or coating, an acrylamide gel or coating, or a polyacrylamide gel or coating.

22 - 44 . (canceled)

45 . A method for sequencing a template nucleic acid molecule, comprising:
(a) introducing one or more primer-binding sites into the template nucleic acid molecule to generate a primed template nucleic acid molecule; (b) contacting said primed template nucleic acid molecule with a substrate comprising primers immobilized thereon, each primer comprising:
(i) a region complementary to a primer-binding site, and
(ii) a barcode sequence indicative of a physical position of the primer on the substrate;
(c) conducting extension reactions using said primers and template nucleic acid molecule as a template, thereby generating extension products, each extension product comprises (i) sequence of a fragment of the template nucleic acid or complement of the fragment, and (ii) sequence of the barcode sequence or a complement thereof; (d) sequencing said extension products to determine sequences of the fragments or complements thereof and barcode sequences or complements thereof; and (e) assembling the sequences of the fragments using the barcode sequence to thereby determine sequence of the template nucleic acid molecule.

46 . The method of claim 45 , further comprising stretching the nucleic acid molecule prior to step (b).

47 . The method of claim 46 , wherein said stretching is performed by molecular combing, molecular threading, transfer printing, magnetic tweezers, or optical tweezers.

48 - 49 . (canceled)

50 . The method of claim 46 , wherein said stretching is performed in nanochannels.

51 - 52 . (canceled)

53 . The method of claim 45 , wherein said substrate is glass, hydrophobic glass, or a polymer coating.

54 - 55 . (canceled)

Assignees

• Zhou Wei
• Crnogorac Filip
• Mcgall Glenn
• Cao Jian
• Centrillion Tech Holdings Corp

Inventors

• Zhou Wei
• Crnogorac Filip
• Mcgall Glenn
• Cao Jian

Classifications

• C12Q1/6874 Primary

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

• C12Q1/6837

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C12Q2565/514

  characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array · CPC title

• C12Q2525/191

  incorporating an adaptor · CPC title

• C12Q2525/155

  incorporating/generating a new priming site · CPC title