METHODS OF SEQUENCING NUCLEIC ACIDS

• US 20100285970A1
• Filed: 03/31/2010
• Published: 11/11/2010
• Est. Priority Date: 03/31/2009
• Status: Abandoned Application

• Current Assignee: Floyd D. Rose
• Original Assignee: Floyd D. Rose
• Inventors: Rose, Floyd D.
• Application Number:
  US12/751,164
• Publication Number:
  US 20100285970A1
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/514 Mismatch repair protein
• CPC Class Codes: C12Q 2537/113 Heteroduplex formation

Abstract

Disclosed are high-throughput methods for sequencing nucleic acid, which entail identifying the complete set of SNPs in a genome of interest in comparison to a wild type or reference DNA whose sequence is known or substantially known. The methods may also entail use of solid supports containing colonies of amplified nucleic acid fragments e.g., prepared by digesting genomic nucleic acid having substantially known sequence, wherein the sequence of the fragments at each coordinate is known. The supports, per se, and apparati containing them, are also provided.

Claims

1. A method of sequencing nucleic acid, comprising:

a) preparing single stranded fragments of a first nucleic acid having a substantially known sequence, wherein each of the fragments has a substantially known sequence;

b) preparing single stranded fragments of a second nucleic acid having an unknown sequence;

c) contacting the single stranded fragments of a) or amplification products (copies) thereof, and the single stranded fragments of b) or copies thereof under conditions that allow formation of heterohybrid nucleic acid, wherein the heterohybrid nucleic acid comprises perfectly complementary heterohybrid nucleic acid and heterohybrid nucleic acid containing a mismatch;

d) distinguishing formation of heterohybrid nucleic acid containing a mismatch from formation of heterohybrid nucleic acid which is perfectly complementary; and

e) determining sequences of the mismatches in d), thus allowing elucidation of the sequence of the second nucleic acid.

2. The method of claim 1, wherein the single stranded fragments of the first nucleic acid and the single stranded fragments of the second nucleic acid are prepared by reacting the first and second nucleic acids with first and second restrictive endonucleases, which may be the same or different.

3. The method of claim 2, wherein the first and second restrictive endonucleases are the same.

4. The method of claim 1 wherein the single stranded fragments of the first nucleic acid or copies thereof, or wherein the single stranded fragments of the second nucleic acid, or copies thereof, are attached to a solid support.

5. The method of claim 4, wherein the single stranded fragments of the first nucleic acid, or the single stranded fragments of the second nucleic acid are amplified prior to being attached to the solid support.

6. The method of claim 4, wherein the single stranded fragments of the first nucleic acid, or the single stranded fragments of the second nucleic acid are amplified after being attached to the solid support.

7. The method of claim 4, wherein each of the amplified fragments comprises 5′

and 3′

flanking sequences of known sequence that serve as primers.

8. The method of claim 4, wherein copies of the single stranded fragments of the first nucleic acid, or copies of the single stranded fragments of the second nucleic acid, are attached to the solid support in the form of colonies.

9. The method of claim 8, wherein the single stranded fragments of the first nucleic acid, or the single stranded fragments of the second nucleic acid are templates which comprise, at their 5′

end, means for attachment to the solid support, and at their 3′

end, a sequence that hybridizes to a 3′

end of a colony primer, wherein the 5′

end of the colony primer comprises means for attachment to the solid support, and wherein the attachment comprises reacting the templates and the colony primers in the presence of the support such that the 5′

ends of the templates and the colony primers become attached to the solid support, and performing at least one round of nucleic acid amplification reaction on the attached templates, thus creating individual colonies of each of the amplified templates.

10. The method of claim 9, further comprising sequencing at least a portion of the amplified templates in each colony to allow for identification of a particular single stranded fragment contained in each colony.

11. The method of claim 1, wherein the single stranded fragments of the second nucleic acid, or copies thereof, are affixed to a solid support, and wherein the single stranded fragments of the first nucleic acid which contain a single nucleotide polymorphism (SNP) are labeled, such that annealing of the labeled single stranded fragment of the first nucleic acid that contains the SNP to a single stranded fragment of the second nucleic acid indicates the presence of the SNP in the single stranded fragment of the second nucleic acid.

12. The method of claim 1, wherein the single stranded fragments of the first nucleic acid, or copies thereof, or the single stranded fragments of the second nucleic acid, or copies thereof, are attached to a solid support, at a known location thereof, which comprises a coordinate.

13. The method of claim 4, wherein the solid support comprises a glass surface and the single stranded fragments of the first nucleic acid or copies thereof, or the single stranded fragments of the second nucleic acid, or copies thereof, are covalently attached to the glass surface.

14. The method of claim 4, wherein the single stranded fragments of the first or second nucleic acid which are not attached to the solid support are amplified via PCR with at least one detectably labeled PCR primer.

15. The method of claim 1, wherein (d) comprises contacting the heterohydrid nucleic acid formed in (c) with a mismatch nicking protein.

16. The method of claim 15, wherein the mismatch nicking protein comprises an all-type nicking enzyme (ATE).

17. The method of claim 16, wherein the ATE comprises Topoisomerase I.

18. The method of claim 16, wherein the ATE is detectably labeled.

19. The method of claim 1, wherein (e) comprises contacting the heterohydrid nucleic acid of (d) with at least one of a mismatch repair protein, an excision repair protein, a chemical modification reagent, or a chemical cleavage reagent.

20. The method of claim 1, wherein (e) comprises sequences both strands of the heterohybrid nucleic acid at the site of a mismatch.

21. A solid support comprising a plurality of coordinates, wherein each coordinate comprises a cluster of amplified single stranded fragments of a nucleic acid attached to the support at the coordinate, wherein at least a portion of the sequence of the fragments is known.

22. The solid support of claim 21, wherein each of the fragments comprises 5′

and 3′

primers, the sequences of which are known.

23. The solid support of claim 21, wherein the entire sequence of each of the fragments is known.