NONRADIOACTIVE MISMATCH ANALYSIS TO DETECT SMALL MUTATIONS IN HUMAN HYPOXANTHINE-GUANINE PHOSPHORIBOSYL TRANSFERASE CDNA

We have combined a cDNA-driven PCR technique and a nonradioactive chem ical-cleavage mismatch method, followed by a direct sequencing for det ecting small mutations in the human hypoxanthine-guanine phosphoribosy l transferase (HPRT) gene. HPRT cDNA was synthesized by RT-PCR from 1, 000 wild-type or HPRT(-) mutant cells. Wild-type cDNA was hybridized w ith mutant cDNA to form heteroduplexes. The resultant mismatched bases were modified and cleaved by base-specific chemicals, followed by ana lysis by denaturing polyacrylamide gel electrophoresis. Cleaved fragme nts were detected without using radioactive materials. Finally, direct sequencing of the PCR products was performed with a focus on a small limited region indicated by the mismatch analysis (focused sequencing) . In this study, three small mutations in exon-3 of HPRT cDNA were det ected and characterized completely with this system. As compared with the radioactive method, this system was shown to be very simple and ef ficient.