Method for generating a three-dimensional nucleic acid containing matrix

• US 10,494,662 B2
• Filed: 10/18/2017
• Issued: 12/03/2019
• Est. Priority Date: 03/12/2013
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Church, George M., Lee, Je-Hyuk, Terry, Richard C., Daugharthy, Evan R.
• Primary Examiner(s): Thomas, David C
• Application Number:
  US15/786,921
• Publication Number:
  US 20180051322A1
• Time in Patent Office: 776 Days
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

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

Claims

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.