Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods

Abstract

BackgroundSpecific applications and modern technologies, like non-invasive prenatal testing, non-invasive cancer diagnostic and next generation sequencing, are currently in the focus of researchers worldwide. These have common characteristics in use of highly fragmented DNA molecules for analysis. Hence, for the performance of molecular methods, DNA concentration is a crucial parameter; we compared the influence of different levels of DNA fragmentation on the accuracy of DNA concentration measurements.ResultsIn our comparison, the performance of the currently most commonly used methods for DNA concentration measurement (spectrophotometric, fluorometric and qPCR based) were tested on artificially fragmented DNA samples. In our comparison, unfragmented and three specifically fragmented DNA samples were used.According to our results, the level of fragmentation did not influence the accuracy of spectrophotometric measurements of DNA concentration, while other methods, fluorometric as well as qPCR-based, were significantly influenced and a decrease in measured concentration was observed with more intensive DNA fragmentation.ConclusionsOur study has confirmed that the level of fragmentation of DNA has significant impact on accuracy of DNA concentration measurement with two of three mostly used methods (PicoGreen and qPCR). Only spectrophotometric measurement was not influenced by the level of fragmentation, but sensitivity of this method was lowest among the three tested. Therefore if it is possible the DNA quantification should be performed with use of equally fragmented control DNA.

Estimation of age-related DNA degradation from formalin-fixed and paraffin-embedded tissue according to the extraction methods

“…It is reasonable to assume that the DNA extracted by QIA was purified by binding to the silica membrane, whereas the DNA extracted by WAX included more contaminants, such as proteins, due to total tissue extraction. PicoGreen dye is not affected by contaminants, however it is affected by DNA integrity (19)(20)(21). In the present study, it was demonstrated that the DNA extracted by WAX was more fragmented than that extracted by QIA.…”

Comparison of Pre-Analytical FFPE Sample Preparation Methods and Their Impact on Massively Parallel Sequencing in Routine Diagnostics

“…Nevertheless prior to massively parallel sequencing fluorescent dye-based quantification methods are commonly used as they are considered to be the easiest, most reliable and cost effective methods [3]. From our point of view qPCR is the least feasible method as it is time consuming, expensive and not practicable in routine laboratories with a high sample throughput, which is also stated by other studies [13], [15]. Additionally, our study showed that samples with a low amount of amplifiable DNA, determined by qPCR, could still be amplified by a well-established PCR and the DNA sample could still be used for mutation detection.…”

Saliva Sampling of Alcoholic Participants using Three Saliva Collection Methods

“…SYBR Green) or 5′ hydrolysis probes (e.g. Taqman ® ) coupled with real-time quantitative PCR (qPCR) (Robin et al 2016;Sedlackova et al 2013). Further evaluation of the results obtained in this study could be attempted in the future with the use of more sensitive methods as accurate measurement of DNA concentration can be especially crucial for downstream analysis of the DNA sample, especially for next generation sequencing (NGS) or high throughput genotyping studies.…”