DHPLC analysis of adenomatous polyposis coli (APC) mutations using ready-to-use APC plates: simple detection of multiple base pair deletion mutations.
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.
Kim
Il-Jin
IJ
Korean Hereditary Tumor Registry, Cancer Research Institute and Cancer Research Center, Seoul National University, Seoul, Korea.
Kim
Kun
K
Kang
Hio Chung
HC
Jang
Sang-Geun
SG
Park
Jae-Gahb
JG
D011125:Adenomatous Polyposis Coli / Q000235:genetics*; D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics*; D020029:Base Pairing / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004252:DNA Mutational Analysis; D005091:Exons; D017353:Gene Deletion; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D009154:Mutation
D025601:Adenomatous Polyposis Coli Protein