Nucleic acid sequencing and mapping

• US 6,537,757 B1
• Filed: 08/08/2000
• Issued: 03/25/2003
• Est. Priority Date: 03/05/1997
• Status: Expired due to Term

• Current Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Original Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Inventors: Markarov, Vladimir L., Langmore, John P.
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/634,267
• Time in Patent Office: 959 Days
• Field of Search: 435/6, 435/91.2, 536/22.1
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

An improvement over the standard Sanger Method for nucleic acid sequencing is described. The novel method does not require denaturation of double-stranded template; rather, sequencing can be carried out directly on the double-stranded template. Embodiments are described with and without oligonucleotide primers.

Claims

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

a) obtaining a sample comprising DNA molecules, said DNA molecules comprising a nick or gap;

b) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′

-3′

exonuclease activity wherein DNA polymerization comprises the incorporation of dideoxynucleotides;

c) digesting said nick translated DNA molecule;

d) separating the product of said digestion;

e) denaturing the separated product; and

f) separating the denatured product.

2. The method of claim 1, wherein said DNA molecules comprise an adaptor.

3. The method of claim 2, wherein said adaptor comprises said nick or gap.

4. The method of claim 1, wherein said nick translation is performed by a single enzyme comprising DNA polymerization activity and 5′

-3′

exonuclease activity.

5. The method of claim 1, wherein said DNA molecules are derived from a cell.

6. The method of claim 5, wherein said DNA molecules are genomic DNA.

7. The method of claim 5, wherein said cell is a human cell.

8. The method of claim 1, wherein said DNA molecules is derived from a virus.

9. The method of claim 1, wherein said DNA molecule is synthetic.

10. The method of claim 1, wherein said incorporation of said dideoxynucleotide results in termination of said nick translation reaction.

11. The method of claim 1, wherein said nick translated product is digested with one or more restriction enzymes.

12. The method of claim 1, wherein the digested product of step (c) is separated by gel electrophoresis.

13. The method of claim 1, wherein said digested product is separated by affinity ligation.

14. The method of claim 1, wherein separating the denatured product of step (e) is by gel electrophoresis.

15. The method of claim 1, wherein said dideoxynucleotide comprises ddATP, ddCTP, ddGTP and ddTTP.

16. The method of claim 1, wherein said dideoxynucleotide is labeled.

17. The method of claim 16, wherein said label is a fluorescent tag.

18. The method of claim 15, wherein said ddATP, ddCTP, ddGTP and ddTTP are each labeled with a different fluorescent tag.

19. The method of claim 1, wherein said method is carried out using a microscale device.

20. The method of claim 19, wherein said microscale device is a microfluidic device.