Detection of six single-nucleotide polymorphisms associated with rheumatoid arthritis by a loop-mediated isothermal amplification method and an electrochemical DNA chip.
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.
Nakamura
Naoko
N
Corporate Research and Development Center, Toshiba Corporation, 1, Komukai-Toshiba-cho, Saiwai-ku, Kawasaki, Kanagawa Prefecture 212-8582, Japan. nao.nakamura@toshiba.co.jp
Ito
Keiko
K
Takahashi
Masayoshi
M
Hashimoto
Koji
K
Kawamoto
Manabu
M
Yamanaka
Mariko
M
Taniguchi
Atsuo
A
Kamatani
Naoyuki
N
Gemma
Nobuhiro
N
D001172:Arthritis, Rheumatoid / Q000175:diagnosis / Q000235:genetics*; D015342:DNA Probes; D004563:Electrochemistry; D005838:Genotype; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis; D020641:Polymorphism, Single Nucleotide
D015342:DNA Probes