Method of sequencing DNA
View Patent ↗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, and the Rp isomer of said analogue and/or the degradation products of said analogue are eliminated from the polymerase reaction step.
1. A method of identifying a base at a target position in a sample nucleic acid sequence, said method comprising:
subjecting a primer hybridized to said sample nucleic acid immediately adjacent to the target position, to a DNA polymerase primer extension reaction in the presence of a nucleotide and the nucleotide degrading enzyme apyrase, 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 DNA polymerase reaction and nucleotide degradation step, and
wherein said elimination reduces or avoids the inhibition of the nucleotide degrading enzyme apyrase which is present during or after the DNA 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 dATPαS or ddATPαS 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 enzymatic degradation.
5. A method as claimed in claim 4 wherein alkaline phosphatase is included in or added to the DNA polymerase reaction mixture.
6. A method as claimed in claim 2 wherein alkaline phosphatase is included in or added to the DNA polymerase reaction mixture.
7. A method as claimed in claim 1 wherein the primer extension reaction is repeated in the presence of further nucleotides.
8. A method of decreasing the inhibition of apyrase when used in a PPi-based sequencing procedure which uses at least one NTPαS, said method comprising eliminating the Rp isomer of NTPαS and/or the degradation products of said analogue from the DNA polymerase reaction and nucleotide degradation step.
9. A method of decreasing the inhibition of luciferase when used in a PPi-based sequencing procedure which uses at least one NTPαS, said method comprising eliminating the Rp isomer of NTPαS and/or the degradation products of said analogue from the DNA polymerase reaction and nucleotide degradation step.
10. The method as claimed in claim 1 , wherein pyrophosphate (PPi) release is detected using the enzymes luciferase and ATP sulphurylase.