Cargando…

Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies

Bisulfite treatment can be used to ascertain the methylation states of individual cytosines in DNA. Ideally, bisulfite treatment deaminates unmethylated cytosines to uracils, and leaves 5-methylcytosines unchanged. Two types of bisulfite-conversion error occur: inappropriate conversion of 5-methylcy...

Descripción completa

Detalles Bibliográficos
Autores principales: Genereux, Diane P., Johnson, Winslow C., Burden, Alice F., Stöger, Reinhard, Laird, Charles D.
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2602783/
https://www.ncbi.nlm.nih.gov/pubmed/18984622
http://dx.doi.org/10.1093/nar/gkn691
_version_ 1782162534367756288
author Genereux, Diane P.
Johnson, Winslow C.
Burden, Alice F.
Stöger, Reinhard
Laird, Charles D.
author_facet Genereux, Diane P.
Johnson, Winslow C.
Burden, Alice F.
Stöger, Reinhard
Laird, Charles D.
author_sort Genereux, Diane P.
collection PubMed
description Bisulfite treatment can be used to ascertain the methylation states of individual cytosines in DNA. Ideally, bisulfite treatment deaminates unmethylated cytosines to uracils, and leaves 5-methylcytosines unchanged. Two types of bisulfite-conversion error occur: inappropriate conversion of 5-methylcytosine to thymine, and failure to convert unmethylated cytosine to uracil. Conventional bisulfite treatment requires hours of exposure to low-molarity, low-temperature bisulfite (‘LowMT’) and, sometimes, thermal denaturation. An alternate, high-molarity, high-temperature (‘HighMT’) protocol has been reported to accelerate conversion and to reduce inappropriate conversion. We used molecular encoding to obtain validated, individual-molecule data on failed- and inappropriate-conversion frequencies for LowMT and HighMT treatments of both single-stranded and hairpin-linked oligonucleotides. After accounting for bisulfite-independent error, we found that: (i) inappropriate-conversion events accrue predominantly on molecules exposed to bisulfite after they have attained complete or near-complete conversion; (ii) the HighMT treatment is preferable because it yields greater homogeneity among sites and among molecules in conversion rates, and thus yields more reliable data; (iii) different durations of bisulfite treatment will yield data appropriate to address different experimental questions; and (iv) conversion errors can be used to assess the validity of methylation data collected without the benefit of molecular encoding.
format Text
id pubmed-2602783
institution National Center for Biotechnology Information
language English
publishDate 2008
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-26027832009-03-05 Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies Genereux, Diane P. Johnson, Winslow C. Burden, Alice F. Stöger, Reinhard Laird, Charles D. Nucleic Acids Res Methods Online Bisulfite treatment can be used to ascertain the methylation states of individual cytosines in DNA. Ideally, bisulfite treatment deaminates unmethylated cytosines to uracils, and leaves 5-methylcytosines unchanged. Two types of bisulfite-conversion error occur: inappropriate conversion of 5-methylcytosine to thymine, and failure to convert unmethylated cytosine to uracil. Conventional bisulfite treatment requires hours of exposure to low-molarity, low-temperature bisulfite (‘LowMT’) and, sometimes, thermal denaturation. An alternate, high-molarity, high-temperature (‘HighMT’) protocol has been reported to accelerate conversion and to reduce inappropriate conversion. We used molecular encoding to obtain validated, individual-molecule data on failed- and inappropriate-conversion frequencies for LowMT and HighMT treatments of both single-stranded and hairpin-linked oligonucleotides. After accounting for bisulfite-independent error, we found that: (i) inappropriate-conversion events accrue predominantly on molecules exposed to bisulfite after they have attained complete or near-complete conversion; (ii) the HighMT treatment is preferable because it yields greater homogeneity among sites and among molecules in conversion rates, and thus yields more reliable data; (iii) different durations of bisulfite treatment will yield data appropriate to address different experimental questions; and (iv) conversion errors can be used to assess the validity of methylation data collected without the benefit of molecular encoding. Oxford University Press 2008-12 2008-11-04 /pmc/articles/PMC2602783/ /pubmed/18984622 http://dx.doi.org/10.1093/nar/gkn691 Text en © 2008 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Genereux, Diane P.
Johnson, Winslow C.
Burden, Alice F.
Stöger, Reinhard
Laird, Charles D.
Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies
title Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies
title_full Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies
title_fullStr Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies
title_full_unstemmed Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies
title_short Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies
title_sort errors in the bisulfite conversion of dna: modulating inappropriate- and failed-conversion frequencies
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2602783/
https://www.ncbi.nlm.nih.gov/pubmed/18984622
http://dx.doi.org/10.1093/nar/gkn691
work_keys_str_mv AT genereuxdianep errorsinthebisulfiteconversionofdnamodulatinginappropriateandfailedconversionfrequencies
AT johnsonwinslowc errorsinthebisulfiteconversionofdnamodulatinginappropriateandfailedconversionfrequencies
AT burdenalicef errorsinthebisulfiteconversionofdnamodulatinginappropriateandfailedconversionfrequencies
AT stogerreinhard errorsinthebisulfiteconversionofdnamodulatinginappropriateandfailedconversionfrequencies
AT lairdcharlesd errorsinthebisulfiteconversionofdnamodulatinginappropriateandfailedconversionfrequencies