%0 Journal Article
%T High recovery of cell-free methylated DNA based on a rapid bisulfite-treatment protocol
%A Inge Pedersen
%A Henrik Krarup
%A Ole Thorlacius-Ussing
%A Poul Madsen
%J BMC Molecular Biology
%D 2012
%I BioMed Central
%R 10.1186/1471-2199-13-12
%X Here we introduce an alternative method for the crucial steps of bisulfite treatment with high recovery. The method is based on an accelerated deamination step and alkaline desulfonation in combination with magnetic silica purification of DNA, allowing preparation of deaminated DNA from patient samples in less than 2 hours.The method presented here allows low levels of DNA to be easily and reliably analysed, a prerequisite for the clinical usefulness of cell-free methylated DNA detection in plasma.Bisulfite induced modification of nucleic acids was originally described in the 1970s [1-3] and since the emergence of PCR and sequencing based detection methods [4,5] bisulfite treatment has played a pivotal role in the analysis of DNA methylation. In its original form it is a time consuming and labour intensive procedure involving numerous experimental steps: DNA denaturation, treatment with bisulfite for 12-16 hr, desalting and desulfonation with NaOH, and finally neutralisation and desalting. Recently published improvements include an accelerated deamination step, cutting down incubation time from 12-16 hr to 40 min, achieved by the use of a more concentrated bisulfite solution at higher temperatures [6,7]. The accelerated and the conventional methods have been explicitly compared by Genereux et al. [8]. The accelerated method leads to a more homogenous conversion of cytosine both across sites and molecules, conceivably due to facilitation of DNA denaturation in concentrated bisulfite solution at 70ˇăC. Hence, inappropriate conversion of 5-methylcytosine, a result of prolonged bisulfite exposure of molecules with complete conversion of cytosine, is more controllable.In addition to deamination of cytosine, bisulfite also induces chain breakage of DNA [9]. The DNA degradation caused by bisulfite treatment results in DNA fragments of an average length of approximately 600 nucleotides [10]. Real-time PCR based methods rely on amplification of short DNA fragments of 60-150 n
%U http://www.biomedcentral.com/1471-2199/13/12