Author/Editor		Fattorini, Paolo; Marrubini, Giorgio; Bonin, Serena; Bertoglio, Barbara; Grignani, Pierangela; Recchia, Elisa; Pitacco, Paola; Procopio, Francesca; Cantoni, Carolina; Zupanič-Pajnič, Irena
Title		Producing standard damaged DNA samples by heating
Type		članek
Vol. and No.		, št. Vol. 549
Publication year		2018
Volume		str. 107-112
ISSN		0003-2697 - Analytical biochemistry
Language		eng
Abstract		Heat-mediated hydrolysis of DNA is a simple and inexpensive method for producing damaged samples in vitro . Despite heat-mediated DNA hydrolysis is being widely used in forensic and clinical validation procedures, the lack of standardized procedures makes it impossible to compare the intra and inter-laboratory outcomes of the damaging treatments. In this work, a systematic approach to heat induced DNA hydrolysis was performed at 70 ⁰C for 0 - 18 h to test the role both of the hydrolysis buffer and of the experimental conditions. Specifically, a trial DNA sample, resuspended in three different media (ultrapure water, 0.1% DEPC-water and, respectively, TE) was treated both in Eppendorf tubes ( Protocol P) and in Eppendorf tubes provided with screwcaps ( Protocol S ). The results of these comparative tests were assessed by normalization of the qPCR results. DEPC-water increased the degradation of the samples up to about 100 times when compared to the ultrapure water. Conversely, the TE protected the DNA from degradation whose level was about 1700 times lower than in samples treated in ultrapure water. Even the employment of the Protocol S affected the level of degradation, by consistently increasing it (up to about 180 times in DEPC-water). Thus, this comparative approach showed that even seemingly apparently trivial and often underestimated parameters modify the degradation level up to 2 - 3 orders of magnitude. The chemical-physical reasons of these fi ndings are discussed together with the role of potential factors such as enhanced reactivity of CO 2 , ROS, NO x and pressure, which are likely to be involved. Since the intra and inter-laboratory comparison of the outcomes of the hydrolytic procedure is the first step toward its standardization, the normalization of the qPCR data by the UV/qPCR ratio seems to be the simplest and most reliable way to allow this. Finally, the supplying (provided with the commercial qPCR kits) of a DNA sample whose degree of degradation is well documented could be helpful in ISO/IEC 17025 validation proce- dures and in pro fi ciency testing.
Keywords		qPCR degraded samples DNA hydrolysis qPCR degradirani vzorci DNK hidroliza