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.
Conclusion: 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.