Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods
- PDF / 711,774 Bytes
- 8 Pages / 595.276 x 793.701 pts Page_size
- 61 Downloads / 191 Views
Biological Procedures Online
RESEARCH
Open Access
Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods Tatiana Sedlackova1*, Gabriela Repiska1, Peter Celec1,3, Tomas Szemes2,3 and Gabriel Minarik1,3
Abstract Background: Specific 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. Results: In 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. Conclusions: Our 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. Keywords: DNA fragmentation, DNA quantitation, Spectrophotometry, PicoGreen, qPCR
Background Circulating nucleic acids are currently studied as a potential diagnostic marker for oncological diseases as well as in relation to non-invasive prenatal diagnosis. Substantial fragmentation and low concentrations are limiting characteristic features of circulating nucleic acids (cNA). 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]. These molecules are released from apoptotic cells after the programmed * Correspondence: [email protected] 1 Institute of Molecular BioMedicine, Faculty of Medicine, Comenius University, Sasinkova 4, Bratislava 811 08, Slovakia Full list of author information is available at the end of the article
enzymatic cleavage process during apoptosis [2]. On the other hand, the fragment lengths of circulating nucleic acids vary in size in cases of malignant disease, because they are released from apoptotic cells as well as necrotic cells [3,4]. The second mentio
Data Loading...
