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

Title: Single nucleotide extension technology for quantitative site-specific evaluation of metC/C in GC-rich regions
Authors: Kaminsky, ZA
Assadzadeh, AA
Flanagan, J
Petronis, A
Item Type: Journal Article
Abstract: 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.
Issue Date: 31-May-2005
Date of Acceptance: 26-May-2005
URI: http://hdl.handle.net/10044/1/40823
DOI: http://dx.doi.org/10.1093/nar/gni094
ISSN: 1362-4962
Publisher: Oxford University Press
Journal / Book Title: Nucleic Acids Research
Volume: 33
Issue: 10
Copyright Statement: © The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated.
Keywords: Science & Technology
Life Sciences & Biomedicine
Biochemistry & Molecular Biology
BIOCHEMISTRY & MOLECULAR BIOLOGY
DNA-METHYLATION ANALYSIS
IP-RP-HPLC
CPG SITES
MS-SNUPE
PYROSEQUENCING(TM)
QUANTIFICATION
POLYMORPHISMS
UNIVERSAL
ISLANDS
Base Composition
Base Pair Mismatch
Base Sequence
Cytosine
DNA Methylation
DNA Primers
GC Rich Sequence
Humans
Molecular Sequence Data
Oligonucleotides
Polymorphism, Genetic
Sequence Analysis, DNA
Sulfites
Templates, Genetic
Developmental Biology
05 Environmental Sciences
06 Biological Sciences
08 Information And Computing Sciences
Publication Status: Published
Appears in Collections:Division of Surgery
Division of Cancer
Faculty of Medicine



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