title: Non-radioactive mismatch analysis to detect small mutations in human hypoxanthine-guanine phosphoribosyl transferase cDNA.
abstract: We have combined a cDNA-driven PCR technique and a non-radioactive chemical-cleavage mismatch method, followed by a direct sequencing for detecting small mutations in the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene. HPRT cDNA was synthesized by RT-PCR from 1,000 wild-type or HPRT(-) mutant cells. Wild-type cDNA was hybridized with mutant cDNA to form heteroduplexes. The resultant mismatched bases were modified and cleaved by base-specific chemicals, followed by analysis by denaturing polyacrylamide gel electrophoresis. Cleaved fragments 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 detected and characterized completely with this system. As compared with the radioactive method, this system was shown to be very simple and efficient.
authors.affiliation: Institute of Clinical Medicine, University of Tsukuba, Ibaraki.
authors.forename: K
authors.initials: K
authors.lastname: Tsuboi
authors.forename: T
authors.initials: T
authors.lastname: Nose
authors.forename: R T
authors.initials: RT
authors.lastname: Okinaka
authors.forename: D J
authors.initials: DJ
authors.lastname: Chen
mesh_terms: D001483:Base Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000737:chemistry*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction
chemical_list: D018076:DNA, Complementary; D007041:Hypoxanthine Phosphoribosyltransferase