Detection of sequence variations in the adenomatous polyposis coli (APC) gene using denaturing high-performance liquid chromatography

G. Wu, C. Sue Richards, W. Wu, M. Hegde, M. Fawkner, B. Chong, D. Love, L. K. Su, P. Lynch, K. Snow

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

We have evaluated the usefulness of denaturing high performance liquid chromatography (dHPLC) for scanning the adenomatous polyposis coli (APC) gene for point mutations small deletions and insertions. Our assay consists of 28 sets of primers to amplify the 15 exons of the APC gene. All PCR reactions were amplified simultaneously using the same reaction conditions in a 96-well format and then analyzed by dHPLC using empirically determined optimum temperatures for partial fragment denaturation. Previously studied DNA specimens from 47 familial adenomatous polyposis (FAP) patients were analyzed by dHPLC and all mutations were correctly identified and confirmed by sequence analysis. This approach identified a single-base substitution in exon 6 and a 2-bp insertion in exon 15 that initially had not been detected by single-strand conformational polymorphism (SSCP) analysis. A novel mutation in exon 15 of the APC gene 2065deIG (codon 689) that had previously been undetected by the protein truncation test (PTT) was also identified by dHPLC. We present our validation studies of dHPLC technology for APC gene analysis in terms of sensitivity and specificity and compare it to current standard scanning technologies including PTT SSCP and conformational sensitive gel electrophoresis (CSGE).

Original languageEnglish (US)
Pages (from-to)281-290
Number of pages10
JournalGenetic Testing
Volume5
Issue number4
DOIs
StatePublished - Jan 1 2001

ASJC Scopus subject areas

  • Genetics(clinical)

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