Patent ID: 7563572

Claim:
A method for long-range allele-specific haplotyping of a gene in a diploid DNA sample from a single individual, wherein the method comprises: (a) preparing a reaction mixture comprising: (i) a diploid DNA sample containing a gene heterozygous at a first locus and at a second locus, wherein the first locus has a target allele and a variant allele that differ by a single nucleotide polymorphism, and the second locus has two alleles that differ by any polymorphism; (ii) an allele-specific extension primer, wherein the 3′ nucleotide of the allele-specific extension primer is complementary to the target allele at the position of the single nucleotide polymorphism; (iii) a common extension primer having a sequence complementary to a remote locus common to both polynucleotide templates of the target allele and the variant allele, wherein the common extension prior encompasses the alleles of the second locus and has the opposite orientation relative to the first extension primer; (iv) a dNTP mixture; and (v) a polymerase enzyme having 3′→5′ exonuclease activity; (b) amplifying the polynucleotide segment under polymerase chain reaction conditions, wherein the annealing temperature is between about 5° C. and about 11° C. greater than the predicted annealing temperature for the allele-specific extension primer and its complementary sequence, thereby selectively effecting hybridization of the first extension primer to the target allele and extension of the primer, relative to the variant allele; (c) detecting the allele of the second locus in the resulting amplification product.