MULTIPLEX DNA AMPLIFICATION AND SOLID-PHASE DIRECT SEQUENCING FOR MUTATION ANALYSIS AT THE HPRT LOCUS IN CHINESE-HAMSTER CELLS

We report here the development of multiplex in vitro DNA amplification and solid-phase direct exon sequencing for the analysis of mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster cells. 18 representative HPRT-deficient mutants, deriv ed either spontaneously, or after exposure to UV light or ionizing rad iation, were analyzed. All 9 hprt exons were simultaneously amplified via the polymerase chain reaction (PCR) for rapid deletion detection. 5 mutants involve single- or multiple-exon deletions. Altered multiple x PCR patterns were detected in mutants Bsp-040, Bsp-065 and BGR-606. Subsequent direct sequence analysis reveals that Bsp-040 and Bsp-065 c arry a 52-bp and a 13-bp intragenic DNA deletion in exon 3, respective ly. BGR-606 contains a 223-bp insertion accompanied by a 10-bp deletio n of intron sequence within exon 4 fragment. Other subtle DNA alterati ons identified by direct exon sequence analysis include single-base su bstitutions, small deletions and insertions, and RNA splicing mutation s.