Evaluation of Different Oligonucleotide Base Substitutions at CpG Binding sites in Multiplex Bisulfite-PCR sequencing.
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.
Centre for Personalised Nanomedicine, Australian Institute for Nanoengineering and Biotechnology, University of Queensland, Brisbane, Australia.
Jennifer
J
Lu
Centre for Personalised Nanomedicine, Australian Institute for Nanoengineering and Biotechnology, University of Queensland, Brisbane, Australia.
Kelin
K
Ru
Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Melbourne, Victoria, 8006, Australia.|Department of Pathology, University of Melbourne, Parkville, Victoria, 3010, Australia.|Peter MacCallum Cancer Center, Parkville, 3010, Australia.
Ida
I
Candiloro
Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Melbourne, Victoria, 8006, Australia.|Department of Pathology, University of Melbourne, Parkville, Victoria, 3010, Australia.|School of Cancer Medicine, La Trobe University, Bundoora, Victoria, 3084, Australia.|Peter MacCallum Cancer Center, Parkville, 3010, Australia.
Alexander
A
Dobrovic
Centre for Personalised Nanomedicine, Australian Institute for Nanoengineering and Biotechnology, University of Queensland, Brisbane, Australia.
Darren
D
Korbie
Centre for Personalised Nanomedicine, Australian Institute for Nanoengineering and Biotechnology, University of Queensland, Brisbane, Australia.
Matt
M
Trau
D001665:Binding Sites; D019295:Computational Biology / Q000379:methods; D018899:CpG Islands; D019175:DNA Methylation; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D021141:Nucleic Acid Amplification Techniques