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DHPLC analysis of adenomatous polyposis coli (APC) mutations using ready-to-use APC plates: simple detection of multiple base pair deletion mutations

Cited 1 time in Web of Science Cited 2 time in Scopus
Authors
Kim, Il-Jin; Kim, Kun; Kang, Hio Chung; Jang, Sang-Geun; Park, Jae-Gahb
Issue Date
2008-06-17
Publisher
Mary Ann Liebert
Citation
Genet Test. 12(2):295-298
Keywords
Adenomatous Polyposis Coli/*geneticsAdenomatous Polyposis Coli Protein/*geneticsBase Pairing/*geneticsChromatography, High Pressure Liquid/*methodsDNA Mutational AnalysisExons*Gene DeletionGenetic Screening/methodsHumans*Mutation
Abstract
The adenomatous polyposis coli (APC), which is the susceptible gene for familial adenomatous polyposis (FAP) and sporadic colorectal cancer, spans 15 exons. The open reading frame of APC is 8529 bp, which encodes 2843 amino acids. Conventional genetic screening involves extensive time as well as high cost and labor. Thus, we developed a novel APC ready-to-use plate for high-throughput mutational analysis by denaturing high performance liquid chromatography (DHPLC). To prepare the ready-to-use APC plate, all 38 primer pairs and PCR mixtures were aliquoted into individual wells of a 96-well plate, and frozen at -20 degrees C until use. All 38 PCR primers were designed to be amplified at the same temperature (52 degrees C). We examined a total of 27 FAP patient samples with APC germline mutations (17 for multiple bp deletions, 1 for 1 bp deletion, 9 for nonsense mutations) and 50 APC-negative noncarriers. All 17 multiple bp deletion mutations were detected during the initial 50 degrees C running analysis and thus ruled out for further analyses. All other mutations were clearly detected under specific optimized conditions. More than 50% of the APC germline mutations were multiple base pair deletions and efficiently selected by omitting time-consuming partial denaturing conditions.
ISSN
1090-6576 (Print)
Language
English
URI
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18554166

http://hdl.handle.net/10371/67775
DOI
https://doi.org/10.1089/gte.2007.0104

https://doi.org/10.1089/gte.2007.0104
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College of Medicine/School of Medicine (의과대학/대학원)Surgery (외과학전공)Journal Papers (저널논문_외과학전공)
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