Rapid quantification of DNA methylation through dNMP analysis following bisulfite-PCR

We report a novel method for rapid quantification of the degree of DNA methylation of a specific gene. Our method combined bisulfite-mediated PCR and quantification of deoxyribonucleoside monophosphate (dNMP) contents in the PCR product through capillary electrophoresis. A specific bisulfite-PCR product was enzymatically hydrolyzed to dNMP monomers which were quantitatively analyzed through subsequent capillary electrophoresis. PCR following bisulfite treatment converts unmethylated cytosines to thymines while leaving methyl-cytosines unchanged. Then the ratio of cytosine to thymine determined by capillary electrophoresis represents the ratio of methyl-cytosine to cytosine in genomic locus of interest. Pure oligonucleotides with known sequences were processed in parallel as standards for normalization of dNMP peaks in capillary electrophoresis. Sources of quantification uncertainty such as carryovers of dNTPs or primers and incomplete hydrolysis were examined and ruled out. When the method was applied to samples with known methylation levels (by bisulfite-mediated sequencing) as a validation, deviations were within ±5%. After bisulfite-PCR, the analytical procedure can be completed within 1.5 h

Open-Switch Fault Diagnosis Algorithm and Tolerant Control Method of the Three-Phase Three-Level NPC Active Rectifier

This paper proposes a fault diagnosis and tolerant control methods for an open-switch fault caused in a three-phase three-level neutral-point-clamped (NPC) pulse-width modulation (PWM) active rectifier. The open-switch fault in the three-level NPC active rectifier causes a distortion in the input phase current and a ripple in the DC-link capacitor voltage. Therefore, proper fault diagnosis and tolerant control methods are required to prevent additional failures and performance degradation in the rectifier system. This paper conducted a detailed analysis of the effect of the single open-switch fault on the NPC PWM active rectifier and proposed a fault diagnosis method utilizing the DC link voltage and the phase angle of the input grid voltage. Furthermore, this paper proposes a fault-tolerant control method to reduce the effect of the open-switch fault by compensating a distorted reference voltage. The effectiveness of the proposed fault diagnosis and tolerant control methods are verified through experimental results