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Nucleic Acids Research 2005 33(10):e95; doi:10.1093/nar/gni094
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Published online 15 June 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Methods Online

Single nucleotide extension technology for quantitative site-specific evaluation of metC/C in GC-rich regions

Zachary A. Kaminsky1,2, Abbas Assadzadeh1, James Flanagan1 and Arturas Petronis1,2,*

1The Krembil Family Epigenetics Laboratory, Centre for Addiction and Mental Health Toronto, ON, Canada M5T 1R8 2University of Toronto Toronto, ON, Canada

*To whom correspondence should be addressed at The Krembil Family Epigenetics Laboratory, Centre for Addiction and Mental Health, Room 28, 250 College Street, Toronto, ON, Canada M5T 1R8. Tel: +1 416 5358501 4880; Fax: +1 416 979 4666; Email: arturas_petronis{at}camh.net

Received April 25, 2005. Revised May 26, 2005. Accepted May 26, 2005.

The development and use of high throughput technologies for detailed mapping of methylated cytosines (metC) is becoming of increasing importance for the expanding field of epigenetics. The single nucleotide primer extension reaction used for genotyping of single nucleotide polymorphisms has been recently adapted to interrogate the bisulfite modification induced ‘quantitative’ C/T polymorphism that corresponds to metC/C in the native DNA. In this study, we explored the opportunity to investigate C/T (and G/A) ratios using the Applied Biosystems (ABI) SNaPshot technology. The main effort of this study was dedicated to addressing the complexities in the analysis of DNA methylation in GC-rich regions where interrogation of the target cytosine can be confounded by variable degrees of methylation in other cytosines (resulting in variable C/T or G/A ratios after treatment with bisulfite) in the annealing site of the interrogating primer. In our studies, the mismatches of the SNaPshot primer with the target DNA sequence resulted in a biasing effect of up to 70% while these effects decreased as the location of the polymorphic site moved upstream of the target cytosine. We demonstrated that the biasing effect can be corrected with the SNaPshot primers containing degenerative C/T and G/A nucleotides. A series of experiments using various permutations of quantitative C/T and G/A polymorphisms at various positions of the target DNA sequence demonstrated that SNaPshot is able to accurately report cytosine methylation levels with <5% average SD from the true values. Given the relative simplicity of the method and the possibility to multiplex C/T and G/A interrogations, the SNaPshot approach may become a useful tool for large-scale mapping of metC.


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