DNA digestion to deoxyribonucleoside: A simplified one-step procedure

Abstract

We present a simple and inexpensive 'one-step' protocol for the hydrolysis of DNA to deoxyribonucleosides. Unlike the older DNA hydrolysis protocol which is cumbersome and labor intensive, thes new protocol is ideal for high-throughput assays and is suitable automation. Using this protocol we were able to hydrolyze several hundred samples within an 8-hour period. The new protocol is fully compatible with LC-MS/MS and gives similar recoveries for all five major deoxyribonucleosides when compared to the older protocol.The requirement to hydrolyze DNA to deoxyribonucleosides is a common component of several assays. To measure the turnover of DNA [1,2] or cells [3][4][5] in various tissues, DNA can be labeled with isotope-labeled precursor. In such studies, the DNA is extracted, hydrolyzed, and analyzed to determine label incorporation by mass spectrometry. Likewise, in vivo experiments in humans, such as examination of the effect of gene polymorphisms [6] and nutritional status [6,7] on DNA metabolism, have used labeled one-carbon donors to label monocyte DNA. Nucleoside hydrolysates of DNA also have been used in determining the base composition of DNA, including investigation of epigenetic modification such as deoxycytosine methylation [8,9], oxidative damage [10,11], or unique DNA composition [12].One of the most commonly used protocols for digesting DNA is the tri-enzyme protocol devised by Crain [13]. However, a major weakness of the Crain protocol is that the first enzyme in the reaction, nuclease P1 (E.C. 3.1.30.1), only can digest single stranded DNA and has a pH optimum (pH ~5) significantly lower and a reaction temperature significantly higher (~50°C) than the other two enzymes (pH >8 and ~37°C) in the reaction. This leads to an overly complex protocol [Fig 1], whereby the DNA must be boiled to denature (and rapidly cooled to prevent reversion), the pH of the sample is adjusted twice, and the sample undergoes three separate enzyme incubations. These complexities limit the number of samples that can be prepared. For logistical reasons (repetitive manipulations of tubes), we were typically [6] restricted to preparing ~60 samples per day when we used the Crain protocol. Furthermore, the Crain protocol uses high buffer concentrations (e.g., 50-100 mmol/L ammonium bicarbonate, pH 7.9) which can interfere with downstream reactions and assays. Therefore, we devised a simple *Corresponding Author: Email: epq@ufl.edu (E.P. Quinlivan). 1 Abbreviations Used: LC-MS/MS: liquid chromatography -tandem mass spectrometry; H-ESI: heated electrospray ionization; dCyt: deoxycytidine; MdCyt: 5-methyldeoxycytidine; dThy: thymidine; dAdn: deoxyadenosine; dGua: deoxyguanosine Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that dur...

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