Novel Tools for Unbiased Dna Differential Methylation Screening

Abstract

DNA differential methylation screening approaches may be hypothesis driven (preselection of the loci to screen) or unbiased (screening precedes mapping of differentially methylated loci). The latter allow for the identification of sequences demonstrating ‘nonclassical‘ methylation behavior in cancer and, thus, widen our concept of tumor biology. Extensive employment of unbiased screening methods is hampered by the troublesome procedures involved in physically mapping the identified differentially methylated DNA fragments. In this special report, we describe a positive experience of optimizing two screening methods, methylation-sensitive arbitrarily primed PCR and amplification of intermethylated sites, with a special focus on simplification, or even exclusion, of sequencing procedures. With our modifications, unbiased screening of DNA differential methylation acquires an easy-to-use workflow, including sophisticated experimental design, high-resolution analysis and simple genomic mapping of the fragments of interest.

KEYWORDS::

• amplification of intermethylated sites
• capillary electrophoresis
• differential methylation screening
• in silico tool
• methylation-sensitive arbitrarily primed PCR
• virtual imaging

Financial & competing interests disclosure

This research was supported by ‘Friends for an Earlier Breast Cancer Test‘ (USA), the Russian Foundation for Basic Research (grant 08-04-01685-a), the Federal Target Program ‘Scientific and Scientific-Educational Personnel of Innovation Russia‘ of the Federal Science and Innovations Agency (Rosnauka, contract no. 02.740.11.0089) and the Federal Agency for Science and Innovations (President of the Russian Federation Young PhD grant MK-418.2009.7). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

This research was supported by ‘Friends for an Earlier Breast Cancer Test‘ (USA), the Russian Foundation for Basic Research (grant 08-04-01685-a), the Federal Target Program ‘Scientific and Scientific-Educational Personnel of Innovation Russia‘ of the Federal Science and Innovations Agency (Rosnauka, contract no. 02.740.11.0089) and the Federal Agency for Science and Innovations (President of the Russian Federation Young PhD grant MK-418.2009.7). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.