Miniaturized lab-on-a-chip (LOC) systems have been developed for genetic and epigenetic analyses in clinical applications because of advantages such as reduced sample size and reagent consumption, rapid processing speed, simplicity, and enhanced sensitivity. Despite tremendous efforts made towards developing LOC systems for use in the clinical setting, the development of LOC systems to analyze DNA methylation, which is an emerging epigenetic marker causing the abnormal silencing of genes including tumor suppressor genes, is still challenging because of the gold standard methods involving a bisulfite conversion step. Existing bisulfite conversion-based techniques are not suitable for clinical use due to their long processing time, labor intensiveness, and the purification steps involved. Here, we present a lab-on-a-chip system for DNA methylation analysis based on bisulfite conversion (LoMA-B), which couples a sample pre-processing module for on-chip bisulfite conversion and a label-free, real-time detection module for rapid analysis of DNA methylation status using an isothermal DNA amplification/detection technique. The methylation status of the RARβ gene in human genomic DNA extracted from MCF-7 cells was analyzed by the LoMA-B system within 80 min (except 16 h for sensor preparation) compared to conventional MS-PCR within 24 h. Furthermore, the LoMA-B system is highly sensitive and can detect as little as 1% methylated DNA in a methylated/unmethylated cell mixture. Therefore, the LoMA-B system is an efficient diagnostic tool for the simple, versatile, and quantitative evaluation of DNA methylation patterns for clinical applications.

LoMA-B: a simple and versatile lab-on-a-chip system based on single-channel bisulfite conversion for DNA methylation analysis

Uterine ERα epigenetic modifications are induced by the endocrine disruptor endosulfan in female rats with impaired fertility.

Monitoring temperature with fluorescence during real-time PCR and melting analysis.

Digital polymerase chain reaction (dPCR) is potentially a primary method for quantifying target DNA regions in a background of nontarget material and is independent of external calibrators. Accurate dPCR measurements require single-molecule detection by conventional PCR assays that may be subject to bias due to inhibition, interference, or sequence-derived PCR inefficiency. Elimination or control of such biases is essential for validation of PCR assays, but this may require a substantial investment in resources. Here we present a mechanism for DNA quantification that does not require PCR assay validation in situations where target DNA quantity is high enough to be measured by physical techniques such as quantitative high-performance liquid chromatography (HPLC) or electrophoresis. A commercially available DNA marker derived from pUC19 was quantified by dPCR and was then used to calibrate an HPLC measuring system for quantifying a DNA amplicon that had a high content of guanidine and cytidine. The dPCR-calibrated HPLC measurement was verified by independent measurement using isotope dilution mass spectrometry (IDMS). HPLC quantification, calibrated with dPCR or IDMS measured DNA markers, provides an effective method for certifying the quantity of genetic reference materials that may be difficult to analyze by PCR. These secondary reference materials may then be used to validate and calibrate quantitative PCR measurements and thus could expand the breadth of applications for which traceability to the International System of Units is possible.

Digital polymerase chain reaction measured pUC19 marker as calibrant for HPLC measurement of DNA quantity.

Real-time quantitative polymerase chain reaction (qPCR) data are found to display periodic patterns in the fluorescence intensity as a function of sample number for fixed cycle number. This behavior is seen for technical replicate datasets recorded on several different commercial instruments; it occurs in the baseline region and typically increases with increasing cycle number in the growth and plateau regions. Autocorrelation analysis reveals periodicities of 12 for 96-well systems and 24 for a 384-well system, indicating a correlation with block architecture. Passive dye experiments show that the effect may be from optical detector bias. Importantly, the signal periodicity manifests as periodicity in quantification cycle (Cq) values when these are estimated by the widely applied fixed threshold approach, but not when scale-insensitive markers like first- and second-derivative maxima are used. Accordingly, any scale variability in the growth curves will lead to bias in constant-threshold-based Cqs, making it mandatory that workers should either use scale-insensitive Cqs or normalize their growth curves to constant amplitude before applying the constant threshold method.

/pdf/system-specific-periodicity-in-quantitative-real-time-4d7x0ield9.pdf

System-specific periodicity in quantitative real-time polymerase chain reaction data questions threshold-based quantitation.

DNA methylation analysis currently requires complex multistep procedures based on bisulfite conversion of unmethylated cytosines or on methylation-sensitive endonucleases. To facilitate DNA methylation analysis, we have developed a quantitative 1-step assay for DNA methylation analysis.

/pdf/quantitative-1-step-dna-methylation-analysis-with-native-3pbd8ugi80.pdf

Quantitative 1-Step DNA Methylation Analysis with Native Genomic DNA as Template

Detecting and resolving position-dependent temperature effects in real-time quantitative polymerase chain reaction.

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Quantitative 1-Step DNA Methylation Analysis with Native Genomic DNA as Template

Detecting and resolving position-dependent temperature effects in real-time quantitative polymerase chain reaction.