Cargando…
Assessing alternative base substitutions at primer CpG sites to optimise unbiased PCR amplification of methylated sequences
BACKGROUND: Determining the role of DNA methylation in various biological processes is dependent on the accurate representation of often highly complex patterns. Accurate representation is dependent on unbiased PCR amplification post bisulfite modification, regardless of methylation status of any gi...
Autores principales: | , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2017
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5379501/ https://www.ncbi.nlm.nih.gov/pubmed/28392841 http://dx.doi.org/10.1186/s13148-017-0328-4 |
_version_ | 1782519618864152576 |
---|---|
author | Candiloro, Ida L. M. Mikeska, Thomas Dobrovic, Alexander |
author_facet | Candiloro, Ida L. M. Mikeska, Thomas Dobrovic, Alexander |
author_sort | Candiloro, Ida L. M. |
collection | PubMed |
description | BACKGROUND: Determining the role of DNA methylation in various biological processes is dependent on the accurate representation of often highly complex patterns. Accurate representation is dependent on unbiased PCR amplification post bisulfite modification, regardless of methylation status of any given epiallele. This is highly dependent on primer design. Particular difficulties are raised by the analysis of CpG-rich regions, which are the usual regions of interest. Here, it is often difficult or impossible to avoid placing primers in CpG-free regions, particularly if one wants to target a specific part of a CpG-rich region. This can cause biased amplification of methylated sequences if the C is placed at those positions or to unmethylated sequences if a T is placed at those positions. METHODS: We examined the effect of various base substitutions at the cytosine position of primer CpGs on the representational amplification of templates and also examined the role of the annealing temperature during PCR. These were evaluated using methylation-sensitive high-resolution melting and Pyrosequencing. RESULTS: For a mixture of fully methylated and unmethylated templates, amplification using the C-, C/T (Y-) and inosine-containing primers was biased towards amplification of methylated DNA. The bias towards methylated sequences increased with annealing temperature. Amplification using primers with an A/C/G/T (N) degeneracy at the cytosine positions was not biased at the lowest temperature used but became increasingly biased towards methylated DNA with increased annealing temperature. Using primers matching neither C nor T was in the main unbiased but at the cost of poor PCR amplification efficiency. Primers with abasic sites were also unbiased but could only amplify DNA for one out of the two assays tested. However, with heterogeneous methylation, it appeared that both the primer type and stringency used have a minimal influence on PCR bias. CONCLUSIONS: This is the first comprehensive analysis of base substitutions at CpG sites in primers and their effect on PCR bias for the analysis of DNA methylation. Our findings are relevant to the appropriate design of a wide range of assays, including amplicon-based next-generation sequencing approaches that need to measure DNA methylation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13148-017-0328-4) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5379501 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-53795012017-04-07 Assessing alternative base substitutions at primer CpG sites to optimise unbiased PCR amplification of methylated sequences Candiloro, Ida L. M. Mikeska, Thomas Dobrovic, Alexander Clin Epigenetics Methodology BACKGROUND: Determining the role of DNA methylation in various biological processes is dependent on the accurate representation of often highly complex patterns. Accurate representation is dependent on unbiased PCR amplification post bisulfite modification, regardless of methylation status of any given epiallele. This is highly dependent on primer design. Particular difficulties are raised by the analysis of CpG-rich regions, which are the usual regions of interest. Here, it is often difficult or impossible to avoid placing primers in CpG-free regions, particularly if one wants to target a specific part of a CpG-rich region. This can cause biased amplification of methylated sequences if the C is placed at those positions or to unmethylated sequences if a T is placed at those positions. METHODS: We examined the effect of various base substitutions at the cytosine position of primer CpGs on the representational amplification of templates and also examined the role of the annealing temperature during PCR. These were evaluated using methylation-sensitive high-resolution melting and Pyrosequencing. RESULTS: For a mixture of fully methylated and unmethylated templates, amplification using the C-, C/T (Y-) and inosine-containing primers was biased towards amplification of methylated DNA. The bias towards methylated sequences increased with annealing temperature. Amplification using primers with an A/C/G/T (N) degeneracy at the cytosine positions was not biased at the lowest temperature used but became increasingly biased towards methylated DNA with increased annealing temperature. Using primers matching neither C nor T was in the main unbiased but at the cost of poor PCR amplification efficiency. Primers with abasic sites were also unbiased but could only amplify DNA for one out of the two assays tested. However, with heterogeneous methylation, it appeared that both the primer type and stringency used have a minimal influence on PCR bias. CONCLUSIONS: This is the first comprehensive analysis of base substitutions at CpG sites in primers and their effect on PCR bias for the analysis of DNA methylation. Our findings are relevant to the appropriate design of a wide range of assays, including amplicon-based next-generation sequencing approaches that need to measure DNA methylation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13148-017-0328-4) contains supplementary material, which is available to authorized users. BioMed Central 2017-04-04 /pmc/articles/PMC5379501/ /pubmed/28392841 http://dx.doi.org/10.1186/s13148-017-0328-4 Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Candiloro, Ida L. M. Mikeska, Thomas Dobrovic, Alexander Assessing alternative base substitutions at primer CpG sites to optimise unbiased PCR amplification of methylated sequences |
title | Assessing alternative base substitutions at primer CpG sites to optimise unbiased PCR amplification of methylated sequences |
title_full | Assessing alternative base substitutions at primer CpG sites to optimise unbiased PCR amplification of methylated sequences |
title_fullStr | Assessing alternative base substitutions at primer CpG sites to optimise unbiased PCR amplification of methylated sequences |
title_full_unstemmed | Assessing alternative base substitutions at primer CpG sites to optimise unbiased PCR amplification of methylated sequences |
title_short | Assessing alternative base substitutions at primer CpG sites to optimise unbiased PCR amplification of methylated sequences |
title_sort | assessing alternative base substitutions at primer cpg sites to optimise unbiased pcr amplification of methylated sequences |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5379501/ https://www.ncbi.nlm.nih.gov/pubmed/28392841 http://dx.doi.org/10.1186/s13148-017-0328-4 |
work_keys_str_mv | AT candiloroidalm assessingalternativebasesubstitutionsatprimercpgsitestooptimiseunbiasedpcramplificationofmethylatedsequences AT mikeskathomas assessingalternativebasesubstitutionsatprimercpgsitestooptimiseunbiasedpcramplificationofmethylatedsequences AT dobrovicalexander assessingalternativebasesubstitutionsatprimercpgsitestooptimiseunbiasedpcramplificationofmethylatedsequences |