Method of sequencing DNA

• US 7,648,824 B2
• Filed: 11/26/2008
• Issued: 01/19/2010
• Est. Priority Date: 09/07/2000
• Status: Expired due to Term

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Tallsjo, Annika, Nyren, Pal, Ronaghi, Mostafa
• Primary Examiner(s): CALAMITA, HEATHER
• Application Number:
  US12/323,706
• Publication Number:
  US 20090155801A1
• Time in Patent Office: 419 Days
• Field of Search: None
• US Class Current: 435/6.15
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

The present invention provides a method of identifying a base at a target position in a sample nucleic acid sequence, said method comprising: subjecting a primer hybridised to said sample nucleic acid immediately adjacent to the target position, to a polymerase primer extension reaction in the presence of a nucleotide, whereby the nucleotide will only become incorporated if it is complementary to the base in the target position, and determining whether or not said nucleotide is incorporated by detecting whether PPi is released, the identity of the target base being determined from the identity of any nucleotide incorporated, wherein, where said nucleotide comprises an adenine base, an α-thio triphosphate analogue of said nucleotide is used, ant the Rp isomer of said analogue and/or the degradation products of said analogue are eliminated from the polymerase reaction step.

Claims

1. A kit for use in a method of identifying a base at a target position in a nucleic acid which comprises:

(a) a polymerase

(b) means for detecting pyrophosphate release;

(c) optionally a nucleotide-degrading enzyme;

(d) alkaline phosphatase;

(e) the Sp isomer of an α

-thiotriphosphate analogue of an adenine nucleotide and optionally one or more Sp isomers of thymine, cytosine or guanine nucleotides; and

(f) optionally, a test specific primer which hybridizes to sample nucleic acid so that the target position is in close proximity to the 3′

end of the primer.

3. The kit of claim 1, which comprises the Sp isomer of dATPα

S or ddATPα

S.

4. The kit of claim 1, which comprises a nucleotide-degrading enzyme being apyrase.

6. The kit of claim 1, wherein the polymerase of (a) is DNA polymerase.

8. The kit of claim 1, wherein the means (b) for detecting pyrophosphate release comprises the enzymes luciferase and ATP sulphurylase.

2. A kit for use in a method of identifying a base at a target position in a nucleic acid which comprises:

(a) a polymerase;

(b) means for detecting pyrophosphate release;

(c) optionally a nucleotide-degrading enzyme;

(d) the Sp isomer of an α

-thiotriphosphate analogue of an adenine nucleotide and optionally one or more Sp isomers of thymine, cytosine or guanine nucleotides; and

(e) optionally, a test specific primer which hybridizes to sample nucleic acid DNA so that the target position is in close proximity to the 3′

end of the primer;

(f) optionally, one or more unmodified thymine, cytosine or guanine nucleotides;

(g) optionally, alkaline phosphatase.

5. The kit of claim 2, which comprises a nucleotide-degrading enzyme being apyrase.

7. The kit of claim 2, wherein the polymerase of (a) is DNA polymerase.

9. The kit of claim 2, wherein the means (b) for detecting pyrophosphate release comprises the enzymes luciferase and ATP sulphurylase.