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

Tatiana Sedlackova,Gabriel Minarik,Peter Celec,Gabriela Repiska,Tomas Szemes

doi:10.1186/1480-9222-15-5

Tatiana Sedlackova, Gabriel Minarik + Show 3 more

Open Access

https://doi.org/10.1186/1480-9222-15-5

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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.

Highlights

Specific applications and modern technologies, like non-invasive prenatal testing, non-invasive cancer diagnostic and generation sequencing, are currently in the focus of researchers worldwide
According to a recent study, the circulating nucleic acids (cNA) are present in the circulation at sizes lower than 1200 bp and most of cNA molecules are clustered into two peaks, first at approximately 162 bp and second at 340 bp, representing a dominant and a minor peak [1]
The aim of our study was to determine whether the degree of DNA fragmentation affects the measurement of DNA concentration with the three most commonly used methods - spectrophotometry, fluorometry and quantitative real-time PCR (qPCR)

Summary

Introduction

Specific applications and modern technologies, like non-invasive prenatal testing, non-invasive cancer diagnostic and generation sequencing, are currently in the focus of researchers worldwide. These have common characteristics in use of highly fragmented DNA molecules for analysis. According to a recent study, the cNA are present in the circulation at sizes lower than 1200 bp and most of cNA molecules are clustered into two peaks, first at approximately 162 bp and second at 340 bp, representing a dominant and a minor peak [1]. The concentration of the undiluted samples could not be established because the fluorescence corresponding to the highest point of the standard curve was lower than

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