Homogeneous Assays for Single-Nucleotide Polymorphism Genotyping : Exo-proofreading Assay based on Loop-mediated Isothermal Amplification

概要

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We have developed a new method Proofreading-LAMP (PR-LAMP) for genotyping single-nucleotide polymorphisms (SNPs) in combination with the use of nucleic acid amplification by loop-mediated isothermal amplification (LAMP) and the 3'-5' exonuclease proofreading (exo-proofreading) activity of DNA polymerase. Using as a model a detection system for the SNP (G1951A) found in the human aldehyde dehydrogenase 2 (ALDH2) gene, typing primers with fluorescent-labeled 3' ends, and cloned DNA as a template, consisting of ALDH2^ * 1, ALDH2 ^* 2, or a mixture of ALDH2^ * 1 and ALDH2^ * 2, and 3'-5' exo+ Pwo DNA polymerase were added to a LAMP reaction system, and amplification and the exo-proofreading reaction proceeded at 60℃ for 30 minutes, at the same time as which SNPs were detected by fluorescence polarization (FP). This procedure permitted evaluation by means of a single-step reaction at 60℃ for 30 minutes for ALDH2^ * 1, ALDH2 ^* 1/ * 2, and ALDH2 ^* 2, and allowed a single nucleotide change to be distinguished with excellent reproducibility. Furthermore, the typing of human genomic samples, similar to the procedure for cloned DNA, yielded results that were in complete agreement with the results of PCR-RFLP. PR-LAMP is accurate, simple, rapid, and robust, and is a flexible method that may be used in applications ranging from point-of-care testing (POCT) to high-throughput screening (HTS).
横浜市立大学の論文
2005-01-31