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Detection of Six Single-Nucleotide Polymorphisms Associated with Rheumatoid Arthritis by a Loop-Mediated Isothermal Amplification Method and an Electrochemical DNA Chip

2007; American Chemical Society; Volume: 79; Issue: 24 Linguagem: Inglês

10.1021/ac0715468

1520-6882

Naoko Nakamura, Keiko Ito, Masayoshi Takahashi, Koji Hashimoto, Manabu Kawamoto, Mariko Yamanaka, Atsuo Taniguchi, Naoyuki Kamatani, Nobuhiro Gemma,

Advanced Biosensing Techniques and Applications

An electrochemical DNA chip using an electrochemically active intercalator and DNA probe immobilized on a gold electrode has been developed for genetic analysis. In this study, the six polymorphisms associated with rheumatoid arthritis (RA), N-acetyltransferase2 (NAT2) gene polymorphisms T341C, G590A, and G857A, methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms C677T and A1298C, and serum amyloid A1 (SAA1) gene promoter polymorphism C-13T were simultaneously detected by the electrochemical DNA chip and the loop-mediated isothermal amplification (LAMP) method, which is a novel technique for DNA amplification. Human genomic DNAs were extracted from blood, and the targets containing the six polymorphisms were amplified by the LAMP method. A sample containing the six LAMP products was reacted with the electrochemical DNA chip using a DNA detection system that controls hybridization reaction, washing, electrochemical detection, and data analysis automatically. A total of 31 samples were genotyped by this method, and the results were completely consistent with those determined by the polymerase chain reaction−restriction fragment length polymorphism (PCR−RFLP) analysis or the PCR direct sequence analysis. The time required for this method was only 2 h, and operations were very simple. Therefore, this method is expected to contribute to personalized medicine based on genotype.