Method of DNA sequencing

• US 6,828,100 B1
• Filed: 12/04/2001
• Issued: 12/07/2004
• Est. Priority Date: 01/22/1999
• Status: Expired due to Term

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Biotage AB (Biotage)
• Inventors: Ronaghi, Mostafa
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/889,812
• Time in Patent Office: 1,099 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/810, 435/91.21, 435/91.5, 935/17, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2522/10 Nucleic acid binding proteins
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

The present invention relates to a method of identifying a base at a target position in a sample nucleic acid sequence wherein a primer, which hybridizes to the sample nucleic acid immediately adjacent to the target position, is provided and the sample nucleic acid and primer are subjected to a polymerase 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 said incorporation is detected, characterized in that, a single-stranded nucleic acid binding protein is included in the polymerase reaction step.

Claims

1. A method of identifying a base at a target position in a sample nucleic acid sequence, comprising providing a sample nucleic acid and a primer which hybridizes to the sample nucleic acid immediately adjacent to the target position, subjecting the sample nucleic acid and primer to a polymerase 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 detecting said incorporation of the nucleotide by monitoring the release of inorganic pyrophosphate, whereby detection of incorporation of said nucleotide is indicative of identification of a base at a target position that is complementary to said nucleotide, and wherein a single-stranded nucleic acid binding protein is included in the polymerase reaction step, and is added after hybridization of the primer to the sample nucleic acid.

2. A method as claimed in claim 1 wherein the single-stranded nucleic acid binding protein is selected from the group consisting of E. coli single-stranded binding protein (Eco SSB), T4 gene 32 protein (T4 gp32), T7 SSB, coliphage N4 SSB, T4 gene 44/62 protein, adenovirus DNA binding protein (AdDBP or AdSSB) and calf thymus unwinding protein (UP1).

3. A method as claimed in claim 2 wherein the single-stranded nucleic acid binding protein is Eco SSB.

4. A method as claimed in claim 1 wherein the sample nucleic acid is DNA.

5. A method as claimed in claim 1 wherein the single-stranded nucleic acid binding protein binds to the sample nucleic acid.

6. A method as claimed in claim 1 wherein the release of inorganic pyrophosphate is detected using ATP sulphurylase and luciferase.

7. A method as claimed in claim 1 wherein apyrase is present during the polymerase reaction.

8. A method as claimed in claim 1 wherein at least 25 bases in the nucleic acid sample are identified.

9. A kit for use in a method of sequencing-by-synthesis which comprises nucleotides for incorporation, a polymerase, luciferase and a single-stranded nucleic acid binding protein.