Method for generating a three-dimensional nucleic acid containing matrix

Patent metadata

Field	Value
Publication number	US-10494662-B2
Application number	US-201715786921-A
Country	US
Kind code	B2
Filing date	Oct 18, 2017
Priority date	Mar 12, 2013
Publication date	Dec 3, 2019
Grant date	Dec 3, 2019

Abstract

Methods of making a three-dimensional matrix of nucleic acids within a cell is provided.

First claim

What is claimed is:

1. A method for nucleic acid sequence identification, comprising
(a) providing a container comprising a biological sample comprising a first plurality of nucleic acid molecules having a relative three-dimensional spatial relationship that is fixed, wherein said first plurality of nucleic acid molecules is a plurality of ribonucleic acid molecules;
(b) in said container, contacting said biological sample comprising said first plurality of nucleic acid molecules having said relative three-dimensional spatial relationship that is fixed with matrix-forming material to generate a three-dimensional polymer matrix that encapsulates said first plurality of nucleic acid molecules of said biological sample, wherein said three-dimensional polymer matrix is formed of a material separate from said first plurality of nucleic acid molecules;
(c) using said first plurality of nucleic acid molecules to generate a second plurality of nucleic acid molecules immobilized to said three-dimensional polymer matrix, which second plurality of nucleic acid molecules have said relative three-dimensional spatial relationship; and
(d) identifying sequences of said second plurality of nucleic acid molecules while said second plurality of nucleic acid molecules is coupled to said three-dimensional polymer matrix.

2. The method of claim 1 , further comprising generating said three-dimensional polymer matrix by polymerizing or cross-linking said matrix-forming material.

3. The method of claim 2 , further comprising contacting said first plurality of nucleic acid molecules having said relative three-dimensional spatial relationship within said biological sample with said matrix-forming material.

4. The method of claim 3 , further comprising, prior to (a), permeabilizing said biological sample.

5. The method of claim 2 , wherein (b) further comprises using a polymerization inducing catalyst, ultraviolet light, or functional cross-linkers to subject said matrix-forming material to polymerization.

6. The method of claim 1 , further comprising subjecting said first plurality of nucleic acid molecules to nucleic acid amplification to generate said second plurality of nucleic acid molecules within said three-dimensional polymer matrix.

7. The method of claim 6 , wherein said subjecting said first plurality of nucleic acid molecules to said nucleic acid amplification comprises introducing reagents into said three-dimensional polymer matrix.

8. The method of claim 7 , wherein said reagents are introduced into said three-dimensional polymer matrix through pores of said three-dimensional polymer matrix.

9. The method of claim 6 , further comprising subjecting said second plurality of nucleic acid molecules to conditions sufficient to couple said second plurality of nucleic acid molecules to said three-dimensional polymer matrix.

10. The method of claim 9 , wherein said second plurality of nucleic acid molecules couple to said three-dimensional polymer matrix by forming covalent bonds to said three-dimensional polymer matrix.

11. The method of claim 6 , further comprising subjecting said second plurality of nucleic acid molecules to conditions sufficient to cross-link said second plurality of nucleic acid molecules to said three-dimensional polymer matrix.

12. The method of claim 6 , wherein said nucleic acid amplification comprises rolling circle amplification or comprises use of polymerase chain reaction.

13. The method of claim 1 , wherein (d) comprises subjecting said second plurality of nucleic acid molecules or derivatives thereof to sequencing to identify said sequences.

14. The method of claim 13 , wherein said sequencing comprises detecting signals indicative of said sequences.

15. The method of claim 14 , wherein said signals are optical signals.

16. The method of claim 1 , wherein said first plurality of nucleic acid molecules comprises a plurality of transcripts.

17. The method of claim 16 , further comprising, prior to (d), subjecting said plurality of transcripts to reverse transcription to yield complementary deoxyribonucleic acid (cDNA) molecules, wherein said sequences identified in (d) are sequences of said cDNA molecules.

18. The method of claim 1 , wherein said biological sample is a tissue from a subject.

19. The method of claim 1 , wherein said second plurality of nucleic acid molecules comprises a functional moiety that couples said second plurality of nucleic acid molecules to said three-dimensional polymer matrix.

20. The method of claim 19 , wherein said functional moiety covalently couples said second plurality of nucleic acid molecule to said three-dimensional polymer matrix.

21. The method of claim 1 , further comprising contacting said biological sample with a preservation agent.

22. The method of claim 1 , wherein said three-dimensional polymer matrix is optically transparent, and wherein (d) comprises optically imaging said three-dimensional polymer matrix to identify said sequences of said second plurality of nucleic acid molecules.

23. The method of claim 1 , wherein said first plurality of nucleic acid molecules is covalently bound to said three-dimensional polymer matrix.

24. The method of claim 23 , wherein said first plurality of nucleic acid molecules is covalently bound to said three-dimensional polymer matrix via cross-linking.

25. The method of claim 1 , wherein said identifying said sequences of said second plurality of nucleic acid molecule comprises hybridizing probes to said second plurality of nucleic acid molecules or derivatives thereof, wherein said probes comprise detectable labels corresponding to said sequences.

26. The method of claim 1 , wherein said biological sample is a cell.

Assignees

• Harvard College

Inventors

• Church George M
• Lee Je-Hyuk
• Terry Richard C
• Daugharthy Evan R

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6874

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

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title