Allele-specific amplification

LaFramboise T, Weir BA, Zhao X et al. Allele-specific amplification in cancer revealed by SNP array analysis. PLoS ComputBiol 2005 l e65. 

The Allele-Specific Amplification Assay (ASA) assay is based on the fact that Taq polymerase will not initiate amplification from a primer that has a mismatch at the 3 ends. Two primers are designed so that the 3 base of the primer corresponds to the site of the genetic mutation to be tested, with either the normal or the mutant sequence at the 3 base positions. An unknown sample can then be tested for the presence of the mutation by using both the normal and the mutant primers in PCR with a common reverse primer. If the sample contains only normal sequence, a PCR product will only be produced when the normal primer is used, and similarly when the sample contains mutant sequence a product will only result from use of the mutant primer. Like the PCR-restriction enzyme method discussed, the ASA approach has also been applied to the detection of mutations in the CYP2D6 gene (16). 

Bergen A, Wang CY, Nakhai B, Goldman D. Mass allele detection (MAD) of rare 5-HT1A structural variants with allele-specific amplification and electro-chemiluminescent detection. Hum Mutat 1996 7 135-143. 

Polymerase chain reaction is particularly useful for genetic analysis because both amplification and primer specific isolation of gene fragments occur simultaneously. Allele-specific amplification can be employed to detect a single base-pair mutation through the use of a specially designed primer which is complementary to... 

Genotype is scored for each sample based on which of the allele specific amplifications were positive. Specificity is determined by the amplification kinetics of each assay and the differential efficiency of the polymerase in extending 3 matched and 3 mismatched primers. This assay format fails to multiplex and requires significant optimization of each assay, but uses cheap unmodified oligos and relatively cheap instrumentation. 

Hebert,., Cayuela, J.M., Daniel, M.-T.etoi. (1994) Detection of minimal residual disease in acute myelomonocytic leukemia with abnormal eosinophils by nested polymerase chain reaction with allele specific amplification. Blood, 84, 2291-2296. 

PCR)-based allele-specific amplification assay to detect the I1781L mutation in the plastidic ACCase of L. rigidum and A. myosuroides plants, providing a quick and efficient method for monitoring a key resistance mechanism to ACC inhibitors in these species [43, 55]. 

Barta, C. Sasvari-Szekely, M. Guttman, A. Simultaneous analysis of various mutations on the 21-hydroxylase gene by multi-allele specific amplification and capillary gel electrophoresis. J. Chromatogr. A, 1998, 817, 281. 

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