Allele-specific PCR (AS-PCR) also is referred to as amplification refractory mutation system (ARMS), PCR amplification of specific alleles (PASA) and PCR amplification with sequence-specific primers (PCR-SSP). AS-PCR is based on the principle that a 3' mismatch between a PCR primer and the template DNA prevents PCR amplification.22 AS-PCR is especially useful for detection of single nucleotide polymorphisms (SNPs) or mutations. For AS-PCR, target DNA is amplified in two separate and simultaneous reactions. Each reaction contains an allele-specific primer (either normal or mutant) and a second primer common to both reactions. PCR is performed under stringent conditions, to prevent PCR amplification if a mismatch is present. Genotype is based on amplification in either one of the reactions alone (homozy-gous normal or mutant) or both reactions (heterozygous). Detection of the amplicon is by either gel electrophoresis or real-time PCR technology (see below). A disadvantage of AS-PCR is that unsuspected nucleotide polymorphisms or mutations located in the DNA template at or adjacent to the 3' binding site of the primer would prevent amplification, leading to incorrect genotyping.
AS-PCR can detect one mutant allele in the presence of 40 copies of the normal allele. AS-PCR can be combined with M-PCR using multiple allele-specific primers in the same reaction tube. This technique is known as multiplex ARMS, a useful method when a single disease is caused by different mutations in one or more genes. Multiplex PCR-SSP also is commonly used in low-resolution HLA typing, in which multiple primer pairs for HLA loci are used along with control primers that amplify a housekeeping gene to verify that amplifiable DNA is present in each reaction tube.
Examples of Applications of AS-PCR
1. Detection of multiple cystic fibrosis CFTR mutations
2. Detection of alpha-1 antitrypsin deficiency mutations
3. Defection of phenylketonuria mutations
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