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

Abstract

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.

Adapting Enzyme-Free DNA Circuits to the Detection of Loop-Mediated Isothermal Amplification Reactions

“…25,26 However, these methods require relatively expensive reagents and devices, and are problematic for point-of-use or point-of-care diagnostics. Moreover, since there are relatively few loop structures in each concatamer, direct readout of the loops without amplification limits sensitivity.…”

Development of an Automated DNA Detection System Using an Electrochemical DNA Chip Technology

“…Feasibility tests of the newly developed automated system Genelyzer TM have already been carried out in laboratories at several universities and very good results were achieved [4], [5]. We applied the previous version system to a determination of three SNPs (C481T G590A G857A) in N-acetyltranferase2 (NAT2) gene testing [4].…”

“…The system was also applied to 6SNP analysis associated with a rheumatoid arthritis (RA) [5]. The six SNPs are three SNPs in NAT2 gene, T341C, G590A and G857A, two SNPs in methylenetetrahydrofolate reductase (MTHFR) gene, C677T and A1298C, and one SNP in serum amyloid A1 (SAA1) gene promoter, C-13T.…”

“…The results for all the samples were clearly classified and all the SNP typing results derived from Genelyzer TM were fully consisted with the results by conventional methods; the PCR-RFLP analysis or the PCR direct sequence analysis. The test condition and the test result in detail are discussed in the reference [5].…”

Ultrasensitive detection of site-specific DNA methylation by loop-mediated isothermal amplification

“…16 However, the sensitivity of these methods is much lower than that of PCR-based methods and is difficult to satisfy the need for detection of DNA methylation in genomic DNA samples. Owing to its high sensitivity and specificity, LAMP has widely been applied to detection of sequence-specific DNA, 22 single nucleotide polymorphisms, 23,24 microRNAs, 25 and pathogenic microorganisms. [17][18][19] However, LCR also requires high precision thermal cycles similar to PCR.…”