Nucleic Acids Research Advance Access originally published online on April 16, 2007
Nucleic Acids Research 2007 35(9):2893-2903; doi:10.1093/nar/gkm055
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Nucleic Acids Research, 2007, Vol. 35, No. 9 2893-2903
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Recovery of bisulfite-converted genomic sequences in the methylation-sensitive QPCR
City of Hope National Medical Center and Beckman Research Institute, 1500 E. Duarte Rd, Duarte, CA 91010, USA
*To whom correspondence should be addressed. Tel: +1 626 301 8316; Fax: +1 626 301 8972; Email: ssmith{at}coh.org
Received December 2, 2006. Revised January 15, 2007. Accepted January 18, 2007.
Many methods for the detection of genomic DNA methylation states have appeared. Currently, nearly all such methods employ bisulfite-mediated deamination of denatured DNA. While this treatment effectively deaminates cytosines to uracils, leaving most 5-methylcytosines intact, it also introduces abasic sites that generate a significant number of single-strand breaks in DNA. We have investigated the interplay of these two processes in order to determine their relative effects on the methylation-sensitive QPCR method. The extent of cleavage of the input DNA is significant and appears to be an increasing function of DNA concentration. Even so, the results suggest that only 
10% of a 62-nt target will be lost due to degradation and targets up to 131 nt will suffer only a 20% loss. More significant losses were found to occur during the subsequent removal of bisulfite and desulfonation steps that appear to be the result of size selectivity associated with matrix binding and elution required prior to QPCR in the most commonly used protocols. For biospecimens yielding <1 µg of DNA, these findings suggest that bisulfite treatment, in current implementations of MS-QPCR, result in low recoveries that preclude reliable analysis of DNA methylation patterns regardless of target size.
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