Evaluation of different oligonucleotide base substitutions at CpG binding sites in multiplex bisulfite-PCR sequencing

Lu, Jennifer, Ru, Kelin, Candiloro, Ida, Dobrovic, Alexander, Korbie, Darren and Trau, Matt (2017) Evaluation of different oligonucleotide base substitutions at CpG binding sites in multiplex bisulfite-PCR sequencing. Scientific Reports, 7 . doi:10.1038/srep45096


Author Lu, Jennifer
Ru, Kelin
Candiloro, Ida
Dobrovic, Alexander
Korbie, Darren
Trau, Matt
Title Evaluation of different oligonucleotide base substitutions at CpG binding sites in multiplex bisulfite-PCR sequencing
Journal name Scientific Reports   Check publisher's open access policy
ISSN 2045-2322
Publication date 2017-03-22
Sub-type Article (original research)
DOI 10.1038/srep45096
Open Access Status DOI
Volume 7
Total pages 10
Place of publication London, United Kingdom
Publisher Nature Publishing Group
Collection year 2018
Language eng
Abstract Multiplex bisulfite-PCR sequencing is a convenient and scalable method for the quantitative determination of the methylation state of target DNA regions. A challenge of this application is the presence of CpGs in the same region where primers are being placed. A common solution to the presence of CpGs within a primer-binding region is to substitute a base degeneracy at the cytosine position. However, the efficacy of different substitutions and the extent to which bias towards methylated or unmethylated templates may occur has never been evaluated in bisulfite multiplex sequencing applications. In response, we examined the performance of four different primer substitutions at the cytosine position of CpG's contained within the PCR primers. In this study, deoxyinosine-, 5-nitroindole-, mixed-base primers and primers with an abasic site were evaluated across a series of methylated controls. Primers that contained mixed- or deoxyinosine- base modifications performed most robustly. Mixed-base primers were further selected to determine the conditions that induce bias towards methylated templates. This identified an optimized set of conditions where the methylated state of bisulfite DNA templates can be accurately assessed using mixed-base primers, and expands the scope of bisulfite resequencing assays when working with challenging templates.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: HERDC Pre-Audit
Australian Institute for Bioengineering and Nanotechnology Publications
 
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