[Detection of alterations of dihydrofolate reductase gene in folate-resistant leukemia cells by in vitro enzymatic amplification].

Three methods for analyzing the products of polymerase chain reaction were applied to detect complex alterations of dihydrofolate reductase (DHFR) gene, in order to assess their value in detection of folate-resistance in leukemia cells. A single point mutation in the second position of codon 31, a T-to-C transition, in trimetrexate (TMQ) resistant MOLT-3/TMQ200 cells was detected by either allele-specific oligonucleotide hybridization or restriction pattern of the PCR product. These two analyses allowed us to detect not only the presence of the mutation, but also the amplification of the mutated gene in TMQ-methotrexate (MTX) doubly-resistant MOLT-3/TMQ200-MTX500 cells. The base change was confirmed by direct sequencing method of the PCR product. Using these analyses of the PCR product, the complex alterations of DHFR gene are to be examined in leukemic patient cells.