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

Research output: Contribution to journalArticle

2 Citations (Scopus)

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
StatePublished - 1995

Fingerprint

Hypoxanthine
Guanine
Transferases
Complementary DNA
Mutation
Polymerase Chain Reaction
Radioactive materials
Electrophoresis
Polyacrylamide Gel Electrophoresis
Exons
Genes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Non-radioactive mismatch analysis to detect small mutations in human hypoxanthine-guanine phosphoribosyl transferase cDNA. / Tsuboi, K.; Nose, T.; Okinaka, R. T.; Chen, D. J.

In: Japanese Journal of Medical Science and Biology, Vol. 48, No. 4, 1995, p. 163-175.

Research output: Contribution to journalArticle

@article{75ddcbb7fa244d889ab706a44b38f81a,
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.",
author = "K. Tsuboi and T. Nose and Okinaka, {R. T.} and Chen, {D. J.}",
year = "1995",
language = "English (US)",
volume = "48",
pages = "163--175",
journal = "Japanese Journal of Infectious Diseases",
issn = "1344-6304",
publisher = "National Institute of Health",
number = "4",

}

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.

PY - 1995

Y1 - 1995

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0028802394&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028802394&partnerID=8YFLogxK

M3 - Article

C2 - 8569042

AN - SCOPUS:0028802394

VL - 48

SP - 163

EP - 175

JO - Japanese Journal of Infectious Diseases

JF - Japanese Journal of Infectious Diseases

SN - 1344-6304

IS - 4

ER -