Method Description

This assay detects the KIT alteration responsible for Asp816Val. The technique used is allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) with fragment analysis on a genetic analyzer. DNA is extracted from bone marrow or blood, and PCR is used to amplify across the alteration site in 2 separate tubes; one contains a reverse primer complementary to the unaltered sequence and the other contains a reverse primer complementary to the altered sequence. Each of these is labeled with a fluorescent tag and contains an identical, non-labeled forward primer. Both primer sets amplify a 200 base pair fragment that differs only at the alteration site. The unaltered fragment should be amplified in all samples. Samples negative for KIT Asp816Val will not have an amplified fragment in the altered sequence reaction tube. Positive samples will have amplified fragments in both tubes. The test gives a qualitative (positive or negative) result only, as the end point PCR used is not reliable for quantification.(Unpublished Mayo method)