45 - Polymerase Chain Reaction Amplification of Specific Alleles: A General Method of Detection of Mutations, Polymorphisms, and Haplotypes

Abstract

The polymerase chain reaction (PCR) utilizes two oligonucleotide primers to amplify a segment of DNA more than 1 million-fold. The PCR can be adapted for the rapid detection of known single-base changes in DNA by using specially designed oligonucleotides in PCR amplification of specific alleles (PASA). The principle of this method is to design an oligonucleotide primer that will preferentially amplify one allele over another. PASA is a generally applicable technique for detection of point mutations or polymorphisms and the presence of small deletions or insertion. But PASA has certain disadvantages that are eliminated by another technique, PCR amplification of multiple specific alleles (PAMSA). This technique allows the rapid detection of more than one allele in a single PCR reaction. PAMSA is useful for detecting mutations and polymorphisms. This chapter discusses general method of detecting haplotypes. PASA has been used to perform population screening, haplotype analysis, patient screening, and carrier testing. Haplotypes are useful in population genetics and medicine. However, determining linkage of haplotypes in the absence of DNA samples from appropriate family members can be difficult and laborious. PASA can be adapted to provide a rapid and reproducible method for haplotyping an individual in the absence of relatives. This method, termed “double PASA,” uses four pairs of allele-specific PCR primers to differentially amplify each of the four possible haplotypes from two biallelic polymorphisms. Double PASA is an important tool for haplotyping doubly heterozygous individuals because the physical linkage of alleles on a strand of DNA is necessary to determine the haplotype.