Non-radioactive mismatch analysis to detect small mutations in human hypoxanthine-guanine phosphoribosyl transferase cDNA

K. Tsuboi, T. Nose, R. T. Okinaka, D. J. Chen

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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.

Original languageEnglish (US)
Pages (from-to)163-175
Number of pages13
JournalJapanese Journal of Medical Science and Biology
Volume48
Issue number4
Publication statusPublished - 1995

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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