Comparative analyses of four different methods of genotyping ALDH2.
BACKGROUND
A number of methods of genotyping single nucleotide polymorphisms (SNPs) are currently available, ranging from the traditional restriction fragment length polymorphism (RFLP) and single-stranded conformational polymorphism (SSCP) to more sophisticated technologies. We determined the utility of three novel methods by genotyping aldehyde dehydrogenase-2 (ALDH2).


METHODS
DNA was isolated from blood samples of 241 control subjects and 74 patients with esophageal cancer. The utility of three novel genotyping methods-melting curve analysis using a LightCycler, SNaPshot analysis using an ABI PRISM 310 genetic analyzer, and denaturing high-performance liquid chromatography using a WAVE DNA fragment analysis system-were compared with that of conventional fluorescent-based polymerase chain reaction (PCR)-SSCP using an ALF express DNA sequencer.


RESULTS
The frequency of the mutant ALDH2*2 allele was significantly higher in patients with esophageal cancer (27.7%) than in control subjects (16.2%; p < 0.01; habitual alcohol drinkers). The melting curve analysis was accurate, more rapid, and easier to use than the SNaPshot analysis or denaturing high-performance liquid chromatography analysis. Fluorescent-based PCR-SSCP proved useful for analyzing a large number of samples.


CONCLUSIONS
Melting curve analysis using the LightCycler is suitable for the genotyping of small numbers of samples in a routine clinical setting; fluorescent-based PCR-SSCP analysis using the ALF express DNA sequencer can be used for large-scale genotyping in epidemiologic studies.
Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan.
Sakae
S
Itoga
Toru
T
Nanmoku
Takayuki
T
Uchimoto
Masahiko
M
Sunaga
Masahiko
M
Nezu
Takeshi
T
Tomonaga
Shoji
S
Harada
Fumio
F
Nomura
D000328:Adult; D000368:Aged; D000444:Aldehyde Dehydrogenase / Q000235:genetics*; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005260:Female; D005821:Genetic Techniques; D005838:Genotype; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational
C096127:ALDH2 protein, human; D000444:Aldehyde Dehydrogenase; D000071396:Aldehyde Dehydrogenase, Mitochondrial