Allele-specific amplification (ASA) allows simultaneous analysis of multiple specific alleles, known point mutations, or known polymorphisms at the same locus. Specificity is achieved in a single PCR reaction by designing one or both PCR primers so that they overlap the site of sequence difference between the amplified alleles.
This results in simultaneous amplification of numerous DNA fragments differing in size, which are subsequently separated by CGE. This technique was applied to the detection of K329E, the most prevalent mutation in medium-chain acyl-coenzyme A dehydrogenase deficiency. The mutant allele produces a DNA fragment that differs in size from the DNA fragment generated by the normal allele. ASA also can be used to detect multiple mutations in a locus.
For example, several point mutations on the 21-hydroxylase gene of a patient with congenital adrenal hyperplasia were analyzed simultaneously by this method. Familial defective apolipoprotein B-100 (FDB) is a dominantly inherited disorder characterized by a decreased affinity of low-density lipoprotein (LDL) for the LDL receptor.
This phenotype is a consequence of a substitution of adenine by guanine in exon 26 of the FDB gene in the region coding for the putative LDL receptor-binding domain of the mature protein. Mutation screening for FDB is performed by ASA followed by CGE. Another mutation that can be detected by ASA and CGE is the G1691A point mutation in the coagulation factor V gene, the most common genetic cause of thrombophilia.
This mutation has been shown to cause resistance to cleavage by activated protein C and is associated with an increased thrombotic risk.
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