METHOD FOR SYNTHESIZING NUCLEIC ACID USING DNA POLYMERASE BETA AND SINGLE MOLECULE SEQUENCING METHOD

• US 20090291440A1
• Filed: 11/16/2006
• Published: 11/26/2009
• Est. Priority Date: 05/24/2006
• Status: Abandoned Application

• Current Assignee: National Institute of Advanced Industrial Science and Technology (Government of Japan), Shimadzu Corporation
• Original Assignee: National Institute of Advanced Industrial Science and Technology (Government of Japan), Shimadzu Corporation
• Inventors: Ishikawa, Mitsuru, Baba, Yoshinobu, Mizushina, Yoshiyuki, Hirano, Ken, Nishimoto, Takahiro
• Application Number:
  US12/301,766
• Publication Number:
  US 20090291440A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• 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 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The present invention provides a nucleic acid synthesis method capable of continuously carrying out an extension reaction and a single molecule sequencing method capable of obtaining base information accurately at high speed. A method for synthesizing a nucleic acid, including the steps of: forming a complex of a target nucleic acid hybridized to a primer oligonucleotide and a DNA polymerase β; allowing the DNA polymerase β to incorporate a fluorescently-labeled dNTP so that the fluorescently-labeled dNTP is bound to the 3′ end of the primer oligonucleotide; and allowing the DNA polymerase β to continuously incorporate fluorescently-labeled dNTP to extend a nucleic acid complementary to the target nucleic acid from the 3′ end of the bound fluorescently-labeled dNTP. A method for sequencing a single nucleic acid molecule, including the steps of said method for synthesizing a nucleic acid, wherein fluorescence emitted from each of the fluorescently-labeled dNTP incorporated into the DNA polymerase β is sequentially detected to carry out the sequencing of the target nucleic acid.

Claims

1. A method for synthesizing a nucleic acid, including the steps of:

forming a complex of a target nucleic acid hybridized to a primer oligonucleotide and a DNA polymerase β

;

allowing the DNA polymerase β

to incorporate a fluorescently-labeled deoxyribonucleotide so that the fluorescently-labeled deoxyribonucleotide is bound to the 3′

end of the primer oligonucleotide; and

allowing the DNA polymerase β

to continuously incorporate fluorescently-labeled deoxyribonucleotides to extend a nucleic acid complementary to the target nucleic acid from the 3′

end of the bound fluorescently-labeled deoxyribonucleotide.

2. The nucleic acid synthesis method according to claim 1, wherein the fluorescently-labeled deoxyribonucleotide is a deoxyribonucleotide labeled with an anionic fluorescent dye.

3. The nucleic acid synthesis method according to claim 2, wherein the anionic fluorescent dye is selected from a group consisting of Alexa Fluor®

488, Alexa Fluor®

532, Alexa Fluor®

546, fluorescein, Oregon Green®

488, Cy3.5, Cy5, Cy5.5, and naphthofluorescein.

4. A method for sequencing a single nucleic acid molecule, including the steps of:

forming a complex of a target nucleic acid to be sequenced hybridized to a primer oligonucleotide and a DNA polymerase β

;

allowing the DNA polymerase β

to incorporate a fluorescently-labeled deoxyribonucleotide so that the fluorescently-labeled deoxyribonucleotide is bound to the 3′

end of the primer oligonucleotide; and

allowing the DNA polymerase β

to continuously incorporate fluorescently-labeled deoxyribonucleotides to extend a nucleic acid complementary to the target nucleic acid to be sequenced from the 3′

end of the bound fluorescently-labeled deoxyribonucleotide, wherein

fluorescence emitted from each of the fluorescently-labeled deoxyribonucleotides incorporated into the DNA polymerase β

is sequentially detected to carry out the sequencing of the target nucleic acid.

5. The method for sequencing a single nucleic acid molecule according to claim 4, wherein two or more kinds of the fluorescently-labeled deoxyribonucleotides are prepared and the two or more kinds of fluorescently-labeled deoxyribonucleotides have different fluorescent labels depending on the kind of their base.

6. The method for sequencing a single nucleic acid molecule according to claim 4, wherein either the target nucleic acid to be sequenced or the DNA polymerase β

is immobilized onto a substrate, and wherein an evanescent field is generated at the surface of the substrate, onto which the target nucleic acid to be sequenced or the DNA polymerase β

has been immobilized, and wherein when the fluorescently-labeled deoxyribonucleotide is incorporated into the DNA polymerase β

, fluorescence emitted from the incorporated fluorescently-labeled deoxyribonucleotide and excited by the evanescent field is detected.

7. The method for sequencing a single nucleic acid molecule according to any one of claim 4, wherein the fluorescently-labeled deoxyribonucleotide is a deoxyribonucleotide labeled with an anionic fluorescent dye.

8. The method for sequencing a single nucleic acid molecule according to claim 7, wherein the anionic fluorescent dye is selected from a group consisting of Alexa Fluor®

488, Alexa Fluor®

532, Alexa Fluor®

546, fluorescein, Oregon Green®

488, Cy3.5, Cy5, Cy5.5, and naphthofluorescein.