Nucleic acid sequencing and mapping

• US 6,117,634 A
• Filed: 03/06/1997
• Issued: 09/12/2000
• Est. Priority Date: 03/05/1997
• Status: Expired due to Fees

• Current Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Original Assignee: University of Michigan
• Inventors: Langmore, John P., Markarov, Vladimir L.
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/811,804
• Time in Patent Office: 1,286 Days
• Field of Search: 435/6, 435/91.2, 935/77, 935/78
• US Class Current: 435/6.11
• 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 nucleic acid, comprising:

a) providing;

i) substantially double-stranded nucleic acid template,

ii) a polymerase having synthetic activity and a 5'"'"'-3'"'"' exonuclease activity,

iii) one or more nucleic acid precursors, and

iv) one or more terminating agents;

b) mixing said polymerase, said one or more precursors, said one or more terminating agents and said template under conditions such that nucleic acid synthesis takes place for a reaction period during which said template remains substantially double-stranded and nucleic acid product is created containing said one or more terminating agents; and

c) detecting said product of said reaction under conditions whereby the nucleic acid sequence of at least a portion of said template is revealed.

2. The method of claim 1, wherein said substantially double-stranded template comprises a single-stranded region.

3. The method of claim 2, wherein a primer is added to said reaction of step (b), said primer capable of hybridizing to said single-stranded region of said substantially double-stranded template.

4. The method of claim 1, wherein prior to step (b) one strand of said substantially double-stranded template is nicked.

5. The method of claim 1, wherein said one or more nucleic acid precursors mixed in step (b) are selected from the group consisting of dATP, dGTP, dTTP and dCTP.

6. The method of claim 1, wherein said one or more terminating agents mixed in step (b) are selected from the group consisting of ddATP, ddGTP, ddTTP and ddCTP.

7. The method of claim 1, wherein said polymerase is Taq DNA polymerase.

8. The method of claim 1, wherein said polymerase is E. coli DNA polymerase I.

9. The method of claim 1, wherein said detecting comprises gel electrophoresis.

10. A method for sequencing nucleic acid, comprising:

a) providing;

i) substantially double-stranded nucleic acid template,

ii) an endonuclease capable of specifically nicking one of the strands of said double-stranded nucleic acid template,

iii) a polymerase having synthetic activity and a 5'"'"'-3'"'"' exonuclease activity,

iv) one or more nucleic acid precursors, and

v) one or more terminating agents;

b) mixing said substantially double-stranded template with said endonuclease under conditions such that a substantially double-stranded template is produced containing a nick on one strand;

c) adding a solution to said nicked template, said solution comprising said polymerase, said one or more precursors, and said one or more terminating agents, whereby said adding is carried out under conditions such that nucleic acid synthesis takes place for a reaction period during which said template remains substantially double-stranded and nucleic acid product is created containing said one or more terminating agents; and

d) detecting said product of said reaction under conditions whereby the nucleic acid sequence of at least a portion of said template is revealed.

11. The method of claim 10, wherein said endonuclease is f1 gpII.

12. The method of claim 10, wherein said one or more nucleic acid precursors mixed in step (b) are selected from the group consisting of dATP, dGTP, dTTP and dCTP.

13. The method of claim 12, wherein one of said one or more nucleic acid precursors are labeled.

14. The method of claim 10, wherein said one or more terminating agents mixed in step (b) are selected from the group consisting of ddATP, ddGTP, ddTTP and ddCTP.

15. The method of claim 14, wherein one of said one ore more terminating agents are labeled.

16. The method of claim 13, wherein said label is selected from the group consisting of radiolabels and fluorescent labels.

17. The method of claim 16, wherein said label is ³² P.

18. The method of claim 17, wherein said detecting comprises gel electrophoresis and autoradiography.

20. The method of claim 16, wherein said template is telomeric DNA.

21. The method of claim 17, wherein said primer comprises the sequence CCCUAA.

19. A method for sequencing nucleic acid, comprising:

a) providing;

i) substantially double-stranded nucleic acid template, said substantially double-stranded template comprising a single-stranded region,

ii) a polymerase having synthetic activity and a 5'"'"'-3'"'"' exonuclease activity,

iii) one or more nucleic acid precursors,

iv) one or more terminating agents, and

v) a primer capable of hybridizing to said single-stranded region of said substantially double-stranded template;

b) mixing said polymerase, said one or more precursors, said one or more terminating agents, said primer and said template under conditions such that nucleic acid synthesis takes place for a reaction period during which said template remains substantially double-stranded and nucleic acid product is created containing said one or more terminating agents; and

c) detecting said product of said reaction under conditions whereby the nucleic acid sequence of at least a portion of said template is revealed.