KIT Asp816Val Mutation Analysis, Qualitative PCR
Method Description Describes how the test is performed and provides a method-specific reference
The KIT mutation assay developed for clinical use in the Mayo Molecular Hematopathology Laboratory detects the KIT mutation responsible for Asp816Val. The technique used is allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) with fragment analysis on an ABI3100 genetic analyzer. Briefly, DNA is extracted from whole bone marrow or blood and PCR is used to amplify across the mutation site in 2 separate tubes; 1 contains a reverse primer complementary to the unmutated sequence and the other contains a reverse primer complementary to the mutated sequence. Each of these reverse primers is labeled with a fluorescent tag and both tubes contain an identical, nonlabeled forward primer. Both primer sets amplify a 200 bp fragment that differs only at the mutation site. The unmutated fragment should be amplified in all samples. Samples negative for KIT Asp816Val will not have an amplified fragment in the mutated reaction tube. Positive samples will have amplified fragments in both the unmutated and mutated 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gives a qualitative (positive or negative) result only, as the end point PCR used is not reliable for quantification.(Unpublished Mayo method)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Friday