DNA sequencing and epigenome analysis

Patent metadata

Field	Value
Publication number	US-10329614-B2
Application number	US-201414909582-A
Country	US
Kind code	B2
Filing date	Aug 1, 2014
Priority date	Aug 2, 2013
Publication date	Jun 25, 2019
Grant date	Jun 25, 2019

Abstract

This disclosure describes, in one aspect, methods for DNA sequencing and performing epigenomic analyses. Generally, the methods include immobilizing a plurality of copies of a DNA molecule on a surface, stretching at least a portion of the immobilized DNA molecules, and sequencing at least a portion of the immobilized, stretched DNA molecules.

First claim

What is claimed is:

1. A method comprising
immobilizing a plurality of copies of a DNA molecule on a surface;
stretching at least a portion of the immobilized DNA molecules;
denaturing at least a portion of the immobilized, stretched DNA molecules;
hybridizing a plurality of probes to at least a portion of the denatured sites of the stretched DNA molecules, thereby creating a hybridized DNA molecule comprising a tag or barcode that identifies the probe on the immobilized DNA,
wherein:
each probe comprises at least five nucleotides complementary to at least five nucleotides of a strand of the denatured site of the stretched DNA molecule; and
a tag or barcode further identifies a location corresponding to the portion of the denatured site of the stretched DNA molecule to which the probe carrying the tag is hybridized;

synthesizing DNA from the probes, wherein the synthesized DNA is complementary to the strand of the stretched DNA to which the probes are hybridized; and
sequencing the DNA synthesized from at least a portion of the probes.

2. The method of claim 1 , wherein the tag or barcode is sequenceable and is read using sequencing by ligation (SBL) with DNA origami.

3. The method of claim 1 , wherein the tag or barcode is sequenceable and is read using sequencing by synthesis (SBS).

4. The method of claim 1 , wherein the tag or barcode is sequenceable and is read using hybridization.

5. The method of claim 1 , wherein the location of one probe is relative to the location of a second probe.

6. The method of claim 1 , wherein the DNA synthesized from the plurality of probes generate overlapping polynucleotide sequences.

7. The method of claim 1 , further comprising removing the elongated probes from the stretched DNA.

8. The method of claim 1 , further comprising determining the polynucleotide sequence of the synthesized DNA of a plurality of elongated probes and the tag or barcode.

9. The method of claim 1 , further comprising using the tag and overlapping polynucleotide sequences to assemble a polynucleotide sequence complementary to the strand of the denatured site of the stretched DNA molecule.

10. The method of claim 1 , further comprising using the tag and non-overlapping polynucleotide sequences to assemble a polynucleotide sequence complementary to the strand of the denatured site of the stretched DNA molecule.

11. The method of claim 1 , further comprising probing the immobilized DNA for an epigenetic modification.

12. The method of claim 11 wherein probing the immobilized DNA for an epigenetic modification comprises using an antibody that specifically binds the epigenetic modification.

13. The method of claim 11 wherein probing the immobilized DNA for an epigenetic modification comprises using a chemical probe that specifically recognizes the epigenetic modification.

14. The method of claim 11 wherein probing the immobilized DNA for an epigenetic modification comprises using a peptide probe that specifically recognizes the epigenetic modification.

15. The method of claim 11 wherein probing the immobilized DNA for an epigenetic modification comprises using an engineered probe that specifically recognizes the epigenetic modification.

16. The method of claim 1 , further comprising probing the immobilized DNA for an epigenetic modification.

17. The method of claim 1 wherein the tag comprises a fluorescent tag.

18. A method comprising
immobilizing a plurality of copies of a DNA molecule on a surface;
stretching at least a portion of the immobilized DNA molecules;
denaturing at least a portion of the immobilized, stretched DNA molecules;
hybridizing a plurality of probes to at least a portion of the denatured sites of the stretched DNA molecules, wherein each probe comprises at least five nucleotides complementary to at least five nucleotides of a strand of the denatured site of the stretched DNA molecule; and
sequencing at least a portion of the immobilized, stretched DNA molecules, wherein sequencing at least a portion of the immobilized, stretched DNA molecules comprises:
synthesizing DNA from the probes, wherein the synthesized DNA is complementary to the strand of the stretched DNA to which the probes are hybridized, thereby creating a plurality of elongated probes;
collecting at least a portion of the elongated probes; and
sequencing the at least a portion of the collected elongated probes.

Assignees

• Stc Unm
• Edwards Jeremy Scott

Inventors

• Edwards Jeremy Scott

Classifications

• C12Q1/6841

  In situ hybridisation · CPC title

• C12Q1/6825 Primary

  Nucleic acid detection involving sensors · CPC title

• C12Q1/6874 Primary

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

• C12Q2523/303

  Applying a physical force on a nucleic acid · CPC title

• C12Q2523/307

  Denaturation or renaturation by electric current/voltage · CPC title