Multiplex Genotyping of Allelic Variants of Genes Involved in Metabolizing Antileukemic Drugs

Resumo

A biochip, primer set, and genotyping protocol were developed to simultaneously address 16 single nucleotide polymorphisms in antileukemic drug metabolism genes, including TPMT, ITPA, MTHFR, SLCO1B1, SLC19A1, NR3C1, GRIA1, ASNS, MTRR, and ABCB1. The genotyping procedure included a one-round multiplex polymerase chain reaction (PCR) with simultaneous incorporation of a fluorescent label into the PCR product and subsequent hybridization on a biochip with immobilized probes. The method was used to test 65 DNA samples of leukemia patients. Fluorescence signal intensity ratios in pairs of wildtype and respective mutant sequence probes were analyzed for all polymorphic markers and demonstrated high accuracy of genotyping. The reliability of genotype determination using the biochip was confirmed by direct Sanger sequencing.