Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation

• US 6,258,568 B1
• Filed: 07/23/1999
• Issued: 07/10/2001
• Est. Priority Date: 12/23/1996
• Status: Expired due to Term

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Pyrosequencing AB
• Inventors: Nyren, Pal
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/331,517
• Time in Patent Office: 718 Days
• Field of Search: 435/91.1, 435/91.2, 435/810
• US Class Current: 435/91.1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The present invention relates to a method of sequencing DNA, based on the detection of base incorporation by the release of pyrophosphate (PPi) and simultaneous enzymatic nucleotide degradation.

Claims

1. A method of identifying a base at a target position in a sample DNA sequence comprising providing a sample DNA sequence and an extension primer, which hybridizes to the sample DNA immediately adjacent to the target position and subjecting the sample DNA and extension primer to a polymerase reaction in the presence of a deoxynucleotide or dideoxynucleotide whereby the deoxynucleotide or dideoxynucleotide will only become incorporated and release pyrophosphate (PPi) if it is complementary to the base in the target position, and detecting any release of PPi enzymically, different deoxynucleotides or dideoxynucleotides being added either to separate aliquots of sample-primer mixture or successively to the sample-primer mixture and subjected to the polymerase reaction to indicate which deoxynucleotide or dideoxynucleotide is incorporated, wherein a nucleotide-degrading enzyme is included during the polymerase reaction step, such that unincorporated nucleotides are degraded, and whereby any release of PPi is indicative of incorporation of deoxynucleotide or dideoxynucleotide and the identification of a base complementary thereto.

2. A method as claimed in claim 1, wherein the nucleotide-degrading enzyme is apyrase.

3. A method as claimed in claim 1, wherein a mixture of nucleotide-degrading enzymes is used having nucleoside triphosphatase, nucleoside diphosphatase and nucleoside monophosphatase activity.

4. A method as claimed in claim 1, wherein the nucleotide-degrading enzyme is immobilised on a solid support.

5. A method as claimed in claim 4, wherein said immobilised nucleotide-degrading enzyme is added after nucleotide incorporation by the polymerase has taken place, and then removed prior to a subsequent nucleotide incorporation reaction step.

6. A method as claimed in claim 1, wherein PPi release is detected using the Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay (ELIDA).

7. A method as claimed in claim 1, wherein the PPi detection enzymes are included in the polymerase reaction step and the polymerase reaction and PPi release detection steps are performed substantially simultaneously.

8. A method as claimed in claim 1, wherein in the polymerase reaction a dATP or ddATP analogue is used which is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for a PPi detection enzyme.

9. A method as claimed in claim 8, wherein the dATP analogue is deoxyadenosine α

-thiotriphosphate (dATPα

S).

10. A method as claimed in claim 1, further comprising the use of the α

-thio analogues of dCTP, dGTP and dTTP.

11. A method as claimed in claim 1, wherein the sample DNA is immobilised or provided with means for attachment to a solid support.

12. A method as claimed in claim 1, wherein the sample DNA is first amplified.

13. A method as claimed in claim 1, wherein the extension primer contains a loop and anneals back on itself and the 3′

end of the sample DNA.

14. A method as claimed in claim 1, wherein an exonuclease deficient (exo⁻

) high fidelity polymerase is used.

15. A method as claimed in claim 1, for identification of a base in a single target position in a DNA sequence comprising subjecting the sample DNA to amplification;

immobilizing the amplified DNA and then subjecting the immobilized DNA to strand separation, removing the non-immobilized strand and providing an extension primer, which hybridizes to the immobilized DNA immediately adjacent to the target position;

subjecting each of four aliquots of the immobilized single stranded DNA to a polymerase reaction in the presence of a dideoxynucleotide, each aliquot using a different dideoxynucleotide whereby only the dideoxynucleotide complementary to the base in the target position becomes incorporated;

subjecting the four aliquots to extension in the presence of all four deoxynucleotides, whereby in each aliquot the DNA which has not reacted with the dideoxynucleotide is extended to form double stranded DNA while the dideoxy-blocked DNA remains as single stranded DNA;

followed by identifying the double stranded and/or single stranded DNA to indicate which dideoxynucleotide was incorporated and hence which base was present in the target position.

16. A kit for use in a method as defined in claim 1, comprising:

(a) a polymerase;

(b) detection enzyme means for identifying pyrophosphate release;

(c) a mixture of nucleotide-degrading enzymes;

(d) deoxynucleotides, or optionally deoxynucleotide analogues, optionally including, in place of dATP, a dATP analogue which is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for a said PPi-detection enzyme; and

(e) optionally a test specific primer which hybridises to sample DNA so that the target position is directly adjacent to the 3′

end of the primer; and

(f) optionally dideoxynucleotides, or optionally dideoxynucleotide analogues, optionally ddATP being replaced by a ddATP analogue which is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for a said PPi-detection enzyme.

17. A method of identifying a base at a target position in a sample DNA sequence comprising arranging a multiplicity of DNA sequences in array format on a solid surface, providing to each sample an extension primer, which hybridizes to the sample DNA immediately adjacent to the target position and subjecting the sample DNA and extension primer to a polymerase reaction in the presence of a deoxynucleotide or dideoxynucleotide whereby the deoxynucleotide or dideoxynucleotide will only become incorporated and release pyrophosphate (PPi) if it is complementary to the base in the target position and detecting any release of PPi enzymically, different deoxynucleotides or dideoxynucleotides being added either to separate aliquots of sample-primer mixture or successively to the same sample-primer mixture and subjected to the polymerase reaction to indicate which deoxynucleotide or dideoxynucleotide is incorporated whereby a nucleotide-degrading enzyme is included during the polymerase reaction step such that unincorporated nucleotides are degraded, and whereby any release of PPi is indicative of incorporation of deoxynucleotide or dideoxynucleotide and the identification of a base complementary thereto.