Non-radioactive mismatch analysis to detect small mutations in human hypoxanthine-guanine phosphoribosyl transferase cDNA.
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.
Tsuboi
K
K
Institute of Clinical Medicine, University of Tsukuba, Ibaraki.
Nose
T
T
Okinaka
R T
RT
Chen
D J
DJ
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
D018076:DNA, Complementary; D007041:Hypoxanthine Phosphoribosyltransferase