Method of sequencing dna

• US 20040142330A1
• Filed: 08/01/2003
• Published: 07/22/2004
• Est. Priority Date: 09/07/2000
• Status: Active Grant

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Ronaghi, Mostafa, Tallsjo, Annika, Nyren, Pal
• Granted Patent: US 7,459,311 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 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, and the Rp isomer of said analogue and/or the degradation products of said analogue are eliminated from the polymerase reaction step.

2. A method as claimed in claim 1 wherein the Rp isomer is eliminated by using a preparation of an α

-thio triphosphate analogue of an adenine nucleotide which contains only the Sp isomer thereof.

3. A method as claimed in claim 2 wherein the Rp isomer is eliminated by using a preparation of dATPα

S or dd ATPα

S which contains only the Sp isomer thereof.

4. A method as claimed in claim 1 wherein the Rp isomer and/or the degradation products of said analogue are eliminated by enzymic degradation.

5. A method as claimed in claim 4 wherein alkaline phosphatase is included in or added to the polymerase reaction mixture.

6. A method as claimed in any preceding claim wherein a nucleotide degrading enzyme is present during or after the polymerase reaction step.

7. A method as claimed in claim 6 wherein the nucleotide degrading enzyme is apyrase.

8. A method as claimed in claim 2 wherein alkaline phosphatase is included in or added to the polymerase reaction mixture.

9. A method as claimed in any one of claims 1 to 8 wherein the primer extension reaction is repeated in the presence of further nucleotides.

10. A method of decreasing the inhibition of apyrase when used in a PPi-based sequencing procedure which uses at least one NTPα

S, said method comprising eliminating the Rp isomer of NTPα

S and/or the degradation products of said analogue from the sequencing reaction.

11. A method of decreasing the inhibition of luciferase when used in a PPi-based sequencing procedure which uses at least one NTPα

S, said method comprising eliminating the Rp isomer of NTPα

S and/or the degradation products of said analogue from the sequencing reaction.

12. 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) one or more nucleotides, preferably deoxynucleotides, including, in place of an adenine nucleotide, an α

-thiotriphoshate analogue of said nucleotide;

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

end of the primer;

13. 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

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

end of the primer;

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

(h) optionally, alkaline phosphatase.