Methods for sequencing nucleic acids

• US 10,597,715 B2
• Filed: 12/05/2014
• Issued: 03/24/2020
• Est. Priority Date: 12/05/2013
• Status: Active Grant

• Current Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Original Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Inventors: Zhou, Wei, Crnogorac, Filip, McGall, Glenn, Cao, Jian
• Primary Examiner(s): Gross, Christopher M
• Application Number:
  US15/100,919
• Publication Number:
  US 20160369334A1
• Time in Patent Office: 1,936 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/515 characterised by the interaction between or sequential use of two or more arrays
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

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.

Claims

1. A method for sequencing a template nucleic acid molecule, comprising:

(a) contacting a stretched and primed template nucleic acid molecule with a substrate, the primed template nucleic acid molecule comprising one or more primer-binding sites, the 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;

(b) conducting extension reactions using the primers and the 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;

(c) sequencing the extension products to determine sequences of the fragments or complements thereof and barcode sequences or complements thereof; and

(d) assembling the sequences of the fragments using the barcode sequence to thereby determine sequence of the template nucleic acid molecule.

2. The method of claim 1, wherein the stretching is performed by molecular combing, molecular threading, transfer printing, magnetic tweezers, or optical tweezers.

3. The method of claim 1, wherein the stretching is performed by molecular combing, by molecular threading, by transfer printing, by magnetic tweezers, by optical tweezers, or in nanochannels.

4. The method of claim 1, wherein the substrate is glass, hydrophobic glass, or a polymer coating.

5. The method of claim 1, wherein the one or more primer-binding sites are introduced into the template nucleic acid molecule via transposon insertion.

6. The method of claim 1, wherein the one or more primer-binding sites are introduced into the template nucleic acid molecule via (i) enzymatic nicking of the template nucleic acid molecule by a nicking enzyme to cleave one strand of the template nucleic acid molecule, (ii) removing 5′

-phosphate from the nicked strand of the templated nucleic acid molecule, and (iii) ligating the primer-binding site to 3′

-end of the nicked strand of the template nucleic acid molecule.

7. The method of claim 1, wherein at least 100 of the extension products are aligned using the barcode sequence indicative of the physical position of the primer on the substrate.

8. The method of claim 1, further comprising, prior to (a):

(a1) covalently bonding initiator species to a surface of the substrate; and

(a2) conducting a surface initiated polymerization of a polymer from the initiator species, wherein the polymer comprises a plurality of polymer chains, wherein a fraction of the plurality of polymer chains comprise the primers.

9. The method of claim 8, wherein the surface is selected from the group consisting of glass, silica, titanium oxide aluminum oxide, indium tin oxide (ITO), silicon, polydimethylsiloxane (PDMS), polystyrene, polycyclic olefins, poly(methyl methacrylate) (PMMA), titanium, and gold.

10. The method of claim 8, wherein in (a2) the conducting the surface initiated polymerization comprises atom-transfer radical polymerization (ATRP).

11. The method of claim 8, wherein in (a2) the conducting the surface initiated polymerization comprises reversible addition fragmentation chain-transfer (RAFT).

12. A method for determining sequence of a template nucleic acid molecule, comprising:

(a) contacting the template nucleic acid molecule with a plurality of probes immobilized on a surface of a nucleic acid probe array, each the probe being uniquely tagged based on its location on the nucleic acid probe array;

(b) performing extension reactions at locations of probe;

template contact points on the template nucleic acid molecule; and

(c) using the tags to compile the sequence of the template nucleic acid molecule, wherein the method further comprises stretching the template nucleic acid molecule prior to (a).

13. The method of claim 12, wherein prior to (b) a plurality of primer-binding sites are incorporated into or attached to the template nucleic acid molecule.

14. The method of claim 13, wherein the plurality of probes are immobilized at different individually addressable locations on the surface, and wherein each probe comprises (i) a binding sequence having sequence complementarity with the primer-binding site, and (ii) a barcode sequence indicative of the addressable location for the probe, wherein the barcode sequence is different for different addressable location.

15. The method of claim 13, wherein the plurality of primer-binding sites are incorporated into or attached to the template nucleic acid on average every at least 200 bp along the template nucleic acid molecule.

16. The method of claim 13, wherein the plurality of primer-binding sites are attached to the template nucleic acid molecule via free primers, each the free primer comprises (i) the primer-binding site, and (ii) a random sequence having complementarity with part of the template nucleic acid molecule, wherein the free primers are not bound to the substrate of the nucleic acid probe array.

17. The method of claim 13, wherein the plurality of primer-binding sites are attached to the template nucleic acid molecule via (i) enzymatic nicking of the template nucleic acid molecule by a nicking enzyme to cleave one strand of the template nucleic acid molecule, (ii) removing 5′

-phosphate from the nicked strand of the templated nucleic acid molecule, and (iii) ligating the primer-binding site to 3′

-end of the nicked strand of the template nucleic acid molecule.

18. The method of claim 12, wherein the template nucleic acid molecule is more than 100,000 base pairs (bp) in length.

19. The method of claim 12, wherein the probes have a density of more than 10⁸/cm².

20. The method of claim 12, wherein the extension reactions produce a plurality of extension products, each the extension product comprising a first complementary sequence of a fragment of the template nucleic acid molecule and a second sequence indicative of the individually addressable location of corresponding probe.