Patent ID: 6503710
Filing Date: 2003-01-07
Classification: C12Q,Y10T

Abstract:
Method for the mass-spectrometric analysis of a known mutation site in genomic DNA wherein the method is performed without purification or isolation of extended product chains during the course of the mass-spectrometric determination comprising:1) providing a DNA segment comprising a genomic sequence including said mutation in site, 2) adding to the DNA, primers comprising linkages that are susceptible to a digestion procedure to remove at least part of the primers and a set of non-terminating nucleotide triphosphates (NTP) and terminating nucleotide triphosphates which form linkages that are not susceptible to said digestion procedure, wherein at least one of the primers or the set of nucleotide triphosphates includes charge tags, 3) hybridizing the primers to the DNA and extending the primers in an enzymatic complementary copy reaction, such that product chains are formed where each product chain includes a section complementary to a DNA portion containing the mutation and one of the charge tags, 4) digesting each of the primers from the extended primer product chains of step 3, such that any of the primer portions no longer attached to the extended product chains are completely degraded, leaving charge tags attached to the remaining extended product chains, 5) charge neutralizing, if not already neutrally charged, the remaining extended product chains except for the charge tags, and 6) mass-spectrometrically determining the mass of the product chains wherein the mass of the extended product chain is used to determine the known mutation.