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Abstract

The VSET method comprises: providing a polynucleotide acid sample comprising at least one target site, and a first region of nucleotides immediately adjacent to the target site; preferably genomic DNA; preferably amplifying the polynucleotide; then combining the polynucleotide sample with: three dideoxynucletides selected from the group of ddGTP, ddATP, ddCTP, and ddTTP; and one deoxynucleotide selected from the group consisting of and dGTP, dATP, dCTP, and dTTP wherein the nucleotide of the deoxynucleotide is not the same as the nucleotide in the dideoxynulceotide; and a mini-sequencing primer complementary to the first region of nucleotides; extending the mini-sequencing primer with a dideoxynucletide or deoxynulceotide whose base is complementary to the base at the target site, to provide extension products; and then identifying the extension products, preferably by (MALDI-TOF) mass spectrometry. One of the three ddNTps is complementary to one of the allelic variations at the single point mutation site while the deoxynucleotide is complementary to the other allelic variant at the single point mutation site. The mini-sequencing primer hybridizes to a polynucleotide sequence immediately next to a single point mutation site and is extended by the DNA polymerases. The genotype at the variable site is determined on the basis of the number of nucleotides contained in the extension products.

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First Claim