DHPLC mutation analysis of the hereditary nonpolyposis colon cancer (HNPCC) genes hMLH1 and hMSH2.
Denaturing high-performance liquid chromatography (DHPLC) is an efficient method for detection of mutations involving a single or few numbers of nucleotides, and it has been successfully used for mutation detection in disease-related genes. Colorectal cancer is one of the most common cancers, and mutations in the genes for hereditary nonpolyposis colon cancer (HNPCC), hMLH1 and hMSH2, also involve mainly point mutations. Sequence analysis is supposed to be a screening method with high sensitivity; however, it is time-consuming and expensive. We therefore decided to test sensitivity and reproducibility of DHPLC for 71 sequence variants in hMLH1 and hMSH2 initially found by sequence analysis in DNA samples of German HNPCC patients. DHPLC conditions of the PCR products were based on the melting pattern of the wild-type sequence of the corresponding PCR fragments. All but one of the 71 mutations was detected using DHPLC (sensitivity of 97%). Running time per sample averaged only 7 min, and the system is highly automated. Thus DHPLC is a rapid and sensitive method for the detection of hMLH1 and hMSH2 sequence variants.
Holinski-Feder
E
E
Department Medical Genetics, University of Munich, Goethestr. 29, 80336, Munich, Germany. elke@pedgen.med.uni-muenchen.de
Müller-Koch
Y
Y
Friedl
W
W
Moeslein
G
G
Keller
G
G
Plaschke
J
J
Ballhausen
W
W
Gross
M
M
Baldwin-Jedele
K
K
Jungck
M
M
Mangold
E
E
Vogelsang
H
H
Schackert
H K
HK
Lohsea
P
P
Murken
J
J
Meitinger
T
T
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods* / Q000706:statistics & numerical data; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D005091:Exons; D014644:Genetic Variation; D006801:Humans; D000070957:MutL Protein Homolog 1; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics*; D009687:Nuclear Proteins; D009691:Nucleic Acid Denaturation; D009857:Oncogenes; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D017931:DNA Primers; D004273:DNA, Neoplasm; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000070957:MutL Protein Homolog 1