DNA sequencing method

• US 5,876,934 A
• Filed: 12/18/1996
• Issued: 03/02/1999
• Est. Priority Date: 12/18/1996
• Status: Expired due to Fees

• Current Assignee: GE Healthcare Bio-Sciences Corporation (General Electric Company)
• Original Assignee: Pharmacia Biotech Inc. (General Electric Company)
• Inventors: Voss, Hartmut, Duthie, Scott, Hultman, Tomas, Sevigny, Pierre
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/768,550
• Time in Patent Office: 804 Days
• Field of Search: 435/6, 435/91.2
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium

Abstract

A method of sequencing a DNA molecule is disclosed. This method comprises the step of exposing the molecule to a mixture of dITP/dGTP in a sequencing elongation reaction whereby compression artifacts are reduced.

Claims

1. A method for determining a nucleotide sequence for a template nucleic acid by a chain termination sequence method comprising the steps of

a) annealing an oligonucleotide primer to a template DNA molecule,

b) adding deoxyadenine-5'"'"'-triphosphate (dATP;

deoxythymine-5'"'"'-triphosphate (dTTP);

a mixture of deoxyguanine-5'"'"'-triphosphate (dGTP) and deoxyinosine-5'"'"'-triphosphate (dITP), and deoxycytosine-5'"'"'-triphosphate (dCTP), or functional analogs thereof, and chain terminators, and a DNA polymerase, wherein a complementary strand synthesis reaction occurs; and

c) analyzing the results of the complementary strand synthesis reaction so that a nucleotide sequence of the template nucleic acid may be obtained.

2. The method of claim 1, wherein the ratio of dITP:

dGTP is between 40;

1 and 1;

5.

3. The method of claim 2, wherein the ratio of dITP:

dGTP is between 20;

1 and 2;

1.

4. The method of claim 1, wherein the ratio of dITP:

dGTP is 5;

1.

5. The method of claim 1, wherein the mixture of dITP and dGTP comprises a mixture of c⁷ dGTP and dITP.

6. The method of claim 1, wherein the polymerase is a thermostable DNA polymerase.

7. The method of claim 4, wherein the polymerase is an N-terminal deleted DNA polymerase.

8. The method according to claim 7, wherein the polymerase is selected from the group consisting of a polymerase having an amino acid sequence as that of Thermus aquaticus DNA polymerase excluding the N-terminal 280 amino acid residues and the F667Y mutation thereof.

9. The method of claim 1 wherein the polymerase is T7 DNA polymerase.

10. The method of claim 1, wherein the sequencing method is according to a cycle sequencing protocol.

11. The method of claim 1, wherein the sequencing method is according to an automated sequencing protocol.

12. The method of claim 1, wherein the template nucleic acid sequence is amplified by polymerase chain reaction.