Method for detecting single nucleotide polymorphisms (SNP's)...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

Reexamination Certificate

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C435S091200, C536S023100

Reexamination Certificate

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06972174

ABSTRACT:
A method of genotyping single nucleotide polymorphisms (“SNP”) and point mutations in nucleic acid based on chain extension by polymerase. This invention is based on the fact that the nucleoside immediately 5′ adjacent to any SNP/point mutation site is known, and the neighboring sequence immediately 3′ adjacent to the site is also known. A primer complementary to the sequence directly adjacent to the SNP on the 3′ side in a target polynucleotide is used for chain elongation. The polymerase reaction mixture contains one chain-terminating nucleotide having a base complementary to the nucleotide directly adjacent to the SNP on the 5′ side in the target polynucleotide. An additional dNTP may be added to produce a primer with the maximum of a two-base extension. The resultant elongation/termination reaction products are analyzed for the length of chain extension of the primer, or for the amount of label incorporation from a labeled form of the terminator nucleotide.

REFERENCES:
patent: 6110709 (2000-08-01), Ausubel et al.
patent: 6479242 (2002-11-01), Guo et al.
patent: WO 93/02212 (1993-02-01), None
patent: WO 00/20853 (2000-04-01), None
Sun et al (Nucleic Acids Research (Jun. 15, 2000) 28(12):e68, p. i-viii).
Okano et al (Sensors and Actuators B (2000) 64:88-94.
Piggee et al (J. Chromatog. A (1997) 781:367-375.
J. Sambrook, et al., Molecular Cloning A Laboratory Manual, Second Edition, 1989, pp. 13.65-13.69, Cold Spring Harbor Laboratory Press.
Ausubel, et al., Current Protocols in Molecular Biology, 1998, pp. 7.4A. 13-7.4A. 19, vol. 1, John Wiley & Sons.

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