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Simple, rapid, and accurate determination of deletion mutations by automated dna sequencing of heteroduplex fragments of the adenomatous polyposis coli ( APC ) gene generated by PCR amplification
Author(s) -
Tamura Kazuo,
Yamamoto Yoshihiro,
Saeki Yoshifumi,
Furuyama Junichi,
Utsunomiya Joji
Publication year - 1993
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.1380020609
Subject(s) - heteroduplex , biology , familial adenomatous polyposis , adenomatous polyposis coli , microbiology and biotechnology , polymerase chain reaction , gene , genetics , dna , mutation , colorectal cancer , cancer
Germline mutations in patients with familial adenomatous polyposis were analyzed by polymerase chain reaction (PCR) amplification of the adenomatous polyposis coli gene. PCR products from heterozygous patients for deletions of this gene formed four distinct bands on polyacrylamide gel electrophoresis. The four fragments were subsequently purified and both strands of each fragment were directly sequenced, using an automated DNA sequencer and the same primers as those for PCR amplification. It was found that the two slower migrating fragments were “bulge” heteroduplexes, while the other two were homoduplexes made up of two wild‐type strands and two deletion‐mutant strands, respectively. The sites of deletions in the adenomatous polyposis coli gene could be exactly determined in four of the five patients. In an attempt to identify deletion‐carriers of familial adenomatous polyposis at the presymptomatic stage, a family study was also carried out, and two children were found to have the same mutations as those of their affected parents. The direct sequencing of heteroduplex fragments generated during PCR amplification is a potentially useful method for detecting mutations of not only the adenomatous polyposis coli gene but also many other genes of genetic diseases. © 1993 Wiley‐Liss, Inc.