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Unit Code 88802:
KIT Asp816Val Mutation Analysis, Qualitative PCR

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Method Description

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 (Unpublished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)

Performing Laboratory Location

Rochester

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