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

Nucleic Acids Res. 2008 Dec;36(22):e150. doi: 10.1093/nar/gkn691. Epub 2008 Nov 4.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 5-Methylcytosine / chemistry*
  • Cytosine / chemistry
  • DNA / chemistry*
  • DNA Methylation*
  • DNA, Single-Stranded / chemistry
  • Nucleic Acid Conformation
  • Oligonucleotides / chemical synthesis
  • Oligonucleotides / chemistry
  • Polymerase Chain Reaction
  • Sequence Analysis, DNA*
  • Sulfites / chemistry*
  • Temperature

Substances

  • DNA, Single-Stranded
  • Oligonucleotides
  • Sulfites
  • 5-Methylcytosine
  • Cytosine
  • DNA
  • hydrogen sulfite