TY - JOUR
T1 - Non-radioactive mismatch analysis to detect small mutations in human hypoxanthine-guanine phosphoribosyl transferase cDNA
AU - Tsuboi, K.
AU - Nose, T.
AU - Okinaka, R. T.
AU - Chen, D. J.
N1 - Copyright:
Copyright 2016 Elsevier B.V., All rights reserved.
PY - 1995
Y1 - 1995
N2 - We have combined a cDNA-driven PCR technique and a nonradioactive 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.
AB - We have combined a cDNA-driven PCR technique and a nonradioactive 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.
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U2 - 10.7883/yoken1952.48.163
DO - 10.7883/yoken1952.48.163
M3 - Article
C2 - 8569042
AN - SCOPUS:0028802394
SN - 0021-5112
VL - 48
SP - 163
EP - 175
JO - Japanese Journal of Medical Science and Biology
JF - Japanese Journal of Medical Science and Biology
IS - 4
ER -