Method for long range allele-specific PCR

A method for determining the molecular haplotype of a gene in a diploid DNA sample, 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;
• (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.

A method for determining the molecular haplotype of the CFTR gene, wherein the method comprises:

• (a) preparing a reaction mixture comprising:
• (i) a diploid DNA sample containing the CFTR gene heterozygous at the R117H locus and heterozygous at the IVS-8 polyT locus;
• (ii) an allele-specific extension primer having a sequence complementary to a region encompassing the R117H locus, and wherein the last 3′ nucleotide is complementary to a target allele selected from the 117R and 117H alleles of the R117H locus;
• (iii) a common extension primer having a sequence complementary to a region common to both alleles of the IVS-8 polyT locus, wherein the common extension primers encompass the IVS-8 polyT locus and have the opposite orientation relative to the allele-specific 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 IVS-8 5T locus in the resulting amplification product.

A kit for determining the molecular haplotype of a gene in 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 polymorphism of one or more nucleotides, and the second locus has two alleles that differ by any polymorphism, wherein the kit comprises:

• (a) an allele-specific extension primer having a sequence complementary at its 3′ nucleotide to the target allele of the first locus;
• (b) a common extension primer having a sequence complementary to a remote locus located a distance of more than 10 kb from the first locus and common to both polynucleotide templates of the target allele and the variant allele, wherein the common extension primer encompasses the alleles of the second locus and has the opposite orientation relative to the allele-specific extension primer;
• (c) a dNTP mixture;
• and
• (d) a polymerase enzyme having 3′→5′ exonuclease activity.