Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies.
Nucleic Acids Res
; 36(22): e150, 2008 Dec.
Article
em En
| MEDLINE
| ID: mdl-18984622
ABSTRACT
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Sulfitos
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DNA
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Análise de Sequência de DNA
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Metilação de DNA
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5-Metilcitosina
Idioma:
En
Ano de publicação:
2008
Tipo de documento:
Article