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
✍ Scribed by Kazuo Tamura; Yoshihiro Yamamoto; Yoshifumi Saeki; Jun-ichi Furuyama; Joji Utsunomiya
- Publisher
- John Wiley and Sons
- Year
- 1993
- Tongue
- English
- Weight
- 628 KB
- Volume
- 2
- Category
- Article
- ISSN
- 1059-7794
No coin nor oath required. For personal study only.
✦ Synopsis
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.