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, the addition of a methyl group to cytosines and adenosines, regulates gene expression on a level that is usually referred to as epigenetic, that is, stably maintained during cell divisions. In humans, aberrant DNA methylation is associated with several malignancies, including cancer and so-called imprinting disorders, making it an attractive target for diagnostic purposes. Here we give a brief introduction to the biology of DNA methylation and present the use of methylation biomarkers in laboratory medicine. DNA methylation assays have become the standard procedure in the diagnosis of imprinting disorders, and they are about to shift cancer diagnostics and prognostics to the next level of molecular medicine. However, there is evidence of problems associated with the introduction of such cancer assays in routine diagnostics. We review several assays that have been proposed for DNA methylation analysis. The assays presented analyse the methylation status of single loci and are based either on a bisulphite-treatment or on methylation-sensitive restriction of the DNA under investigation.

DNA methylation analysis.

Use of droplet digital PCR technology (ddPCR) is expanding rapidly in the diversity of applications and number of users around the world. Access to relatively simple and affordable commercial ddPCR technology has attracted wide interest in use of this technology as a molecular diagnostic tool. For ddPCR to effectively transition to a molecular diagnostic setting requires processes for method validation and verification and demonstration of reproducible instrument performance. In this study, we describe the development and characterization of a DNA reference material (NMI NA008 High GC reference material) comprising a challenging methylated GC-rich DNA template under a novel 96-well microplate format. A scalable process using high precision acoustic dispensing technology was validated to produce the DNA reference material with a certified reference value expressed in amount of DNA molecules per well. An interlaboratory study, conducted using blinded NA008 High GC reference material to assess reproducibilit...

Interlaboratory Reproducibility of Droplet Digital Polymerase Chain Reaction Using a New DNA Reference Material Format.

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

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