Allele-specific PCR

Ruano G, Kidd KK. Direct haplotyping of chromosomal segments from multiple heterozygotes via allele-specific PCR amplification. Nucleic Acids Res 1989 17[20] 8392. 

Allele-specific PCR is another powerful methodology designed to measure minor variants in DNA. PCR primers are designed to specifically amplify one of the two alleles, taking advantage of the greatest dynamic range of qPCR to detect even very minor populations (96). 

Heim HM, Meyer UA. Genotyping of poor metabolisers of debrisoquine by allele-specific PCR amplification. Lancet 1990 336 529-532. 

Figure 16 Haplotyping using MALDI-TOF MS. A schematic representation of Allele-specific PCR coupled with MassCLEAVE is depicted.

The A1555G mutation in the human mitochondrial 12S RNA, which has been associated with hearing loss after aminoglycoside administration (63) and has been rmph-cated in maternally inherited hearing loss in the absence of aminoglycoside exposure in some families, can be identified by a simple and rapid method for large-scale screening that uses one-step multiplex allele-specific PCR (64). 

Blasczyk R, Ritter M, Thiede C, Wehling J, Hintz G, Neubauer A, et al. Simple and rapid detection of factor V Leiden by allele specific PCR amplification. Thromb Haemost 1996 75 757-9. 

In conclusion, a novel bioluminescent pyrophosphate assay utilizing the PPDK-luciferinAuciferase reaction was established in order to measure quantitatively PCR products. Detection of pyrophosphate (1.56x10 mol/assay) was possible with the proposed method. Furthermore, this bioluminescent assay in association with allele-specific PCR was applied to the analysis of the dex gene of mutans streptococcus. The novel bioluminescent assay for PCR product based on the PPDK-luciferin/luciferase reaction appears to afford a suitable technique for diagnosis and prevention of bacterial infection and disease. 

Imamura O, Arakawa H, Maeda M. Simple and rapid bioluminescent detection of two verotoxin genes using allele specific PCR of E.coli 0157 H7. Luminescence 2003 18 107-12. 

Allele specific PCR uses three primers one allele specific primer for each allele with the polymorphic site at the 3 end, and a common reverse primer (Figure 3.2c) [61, 62]. Using a polymerase without 3 -5 proofreading activity, samples are amplified twice, once using each allele specific primer. 

A variety of molecular techniques are available to detect point mutations at a specific hot spot (e.g., the KRAS mutation at codons 12 and 13 or the BRAF mutation at codon 600). These methods include realtime PCR amplification and post-PCR melting curve analysis, allele-specific PCR, direct DNA sequencing, pyrosequencing, and PCR-RFLP. o,31-37 q methods demonstrate reliable detection of point mutations, such as the KRAS mutation in colorectal cancer or the BRAF mutation in thyroid cancer. 

Sapio MR, Posca D, Troncone G, et al. Detection of BRAE mutation in thyroid papillary carcinomas by mutant allele-specific PCR amplification (MASA). Eur J Endocrinol. 2006 154 341-348. 

As an alternative to the allele-specific PCR and the restriction digest of PCR products (often called PCR-RFLP), several commercial platforms are available to genotype individual SNPs in large numbers of samples. In our discussion here, we will focus on three commonly used platforms the 5 -exonuclease TaqMan assay. 

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