[Application of denaturing high performance liquid chromatography to mutation detection of the c-kit gene in...

The aim of study was to detect mutation of tyrosine domain of c-kit gene in mastocytosis using denaturing high performance liquid chromatography technique, and to investigate the significance of this gene mutation in diagnosis and therapy of mastocytosis. Genomic DNA was obtained from bone marrow or peripheral blood leukocytes using the phenol/chloroform method from 7 mastocytosis patients. PCR was performed with AmpliTaq Gold DNA polymerase and 100 ng genomic DNA to amplify the entire coding sequence and exon-intron boundaries of c-kit exon 17 approximately exon 19. Denaturing high-performance liquid chromatography (DHPLC) analysis was performed on a WAVE DNA Fragment Analysis System. Each PCR product was mixed with an equal quantity of amplified human placental DNA (served as normal control) and was denatured at 95 degrees C for 5 minutes, followed by slowly cooling down to room temperature by 1.5 degrees C per minute to allow heteroduplexes formation. All the conditions for the DHPLC analysis, including melting temperature and buffer gradients were determined using the Transgenomic software Navigator. Samples with extra peaks or with different peak form on DHPLC were directly sequenced using the BigDye Terminator Cycle Sequencing Reaction kit and ABI 3100 Genetics Analyser. The results showed that DHPLC revealed an aberrant peak in one patient in exon 17 and the D816V mutation was identified by direct sequencing. The other two patients had an extra peak for exon 18/19 and direct sequencing revealed a conservative sequence change (L862L) within exon 18. It is concluded that denaturing high performance liquid chromatography is a high efficiency and reliable technique for mutation detection of c-kit gene and the detection results would be helpful for the selection of therapy in mastocytosis.