DNA cycle sequencing

• US 5,741,640 A
• Filed: 05/18/1995
• Issued: 04/21/1998
• Est. Priority Date: 09/27/1991
• Status: Expired due to Term

• Current Assignee: GE Healthcare Bio-Sciences Corporation (General Electric Company)
• Original Assignee: Amersham Life Science, Inc. (GE Healthcare Technologies, Inc.)
• Inventors: Fuller, Carl W.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/443,468
• Time in Patent Office: 1,069 Days
• Field of Search: 435/91.2, 435/91.1, 435/6, 435/5, 424/94.1, 536/24.3-24.33
• US Class Current: 435/6.11
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/525 Phosphatase
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators

Abstract

Method for sequencing DNA which includes the following steps: providing a polynucleotide primer complementary to a region of the DNA to be sequenced providing the DNA to be sequenced, and contacting that primer and DNA together in the presence of a DNA polymerase and between 1 and 3 dNTPs, at least one of the dNTPs being labeled. The primer and DNA are contacted under conditions which allow extension of the primer by addition of one or more of the dNTPs to the primer to form an extended primer. The primer and DNA are then dissociated, generally by heating, and the contacting and dissociating steps repeated a plurality of times (usually 10-200 times). Finally, the extended primer is contacted with the DNA in the presence of a DNA polymerase (which is generally the same polymerase as used in the initial labeling step) all four dNTPs and a chain terminating agent.

Claims

1. A method for sequencing DNA, comprising the steps of:

a) contacting a polynucleotide primer complementary to a region of said DNA to be sequenced, and said DNA to be sequenced, together in the presence of a DNA polymerase, and between one and three dNTPs at least one dNTP being labeled to allow extension of said primer by addition of one or more said dNTPs to said primer, to form an extended primer,

b) dissociating said extended primer from said DNA,

c) repeating steps a) and b) a plurality of times, and

d) contacting said extended primer with said DNA to be sequenced in the presence of a DNA polymerase, four dNTPs and a chain terminating agent.

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

3. The method of claim 2, wherein said polymerase is Δ

Taq DNA polymerase.

4. The method of claim 1, wherein said polymerase is chosen from T7 DNA polymerase having a low exonuclease activity, Klenow, AMV reverse transcriptase, Bst DNA polymerase, Tth DNA pollEnerase, Vent polymerase, exo-free vent polymerase, and exo-free Klenow.

5. The method of claim 1, wherein said label is ³⁵ S.

6. The method of claim 1, wherein said label is a fluorescent label.

7. The method of claim 1, wherein said label is a chemiluminescent label.

8. The method of claim 1, wherein said label is a ligand detectable by indirect enzyme-linked assay.

9. The method of claim 1, wherein said plurality is between 10 to 200.

10. The method of claim 1, wherein said method further comprises after step d) a further step e) of separating said further extended primer from said DNA.

11. The method of claim 10, wherein said steps d) and e) are repeated a plurality of times.

12. The method of claim 11, wherein said plurality is between 10 to 200.

13. The method of claim 1, wherein said DNA is M13 DNA.

14. The method of claim 1, wherein said DNA is plasmid DNA.

15. The method of claim 1, wherein said DNA comprises an f1 origin.

16. The method of claim 1, wherein said DNA is lambda DNA.

17. The method of claim 1, wherein said DNA is produced by amplification.

18. The method of claim 17, wherein said amplification is by PCR.