All tags Epigenetics Bisulfite conversion: technical considerations and applications

Bisulfite conversion: technical considerations and applications

Studying DNA methylation presents unique challenges when combined with RNA and DNA sequence-based studies. We discuss technical considerations and the latest applications for bisulfite conversion.

DNA methylation cannot be directly studied using traditional DNA amplification approaches because the marks are not maintained during sample preparation steps. Bisulfite conversion is currently one of the most widely used approaches to convert DNA methylation marks into a suitable template for amplification and downstream analysis.

​​In bisulfite conversion, DNA is treated with NaOH and sodium bisulfite in a chemical reaction that converts cytosine (C) bases into uracil (U), while methylated cytosines (5-mC) are protected from the conversion.

During downstream analysis such as with PCR or sequencing, unmethylated (unprotected) C bases that undergo deamination in the bisulfite reaction will be interpreted as thymine (T), whereas 5-mC bases (protected) will remain unchanged, indicating any locations of DNA methylation (Frommer et al., 1992).

Bisulfite conversion: technical considerations

Bisulfite conversion is a very powerful method because it is relatively simple to perform, and it is capable of delivering single-base resolution of DNA methylation status.

However, the method does have some drawbacks, which should be considered by researchers before embarking on a new study:

  • Incomplete conversion (or on occasion, over-conversion) can occur under sub-optimal reaction conditions leading to insufficient DNA denaturation, or when the DNA strands re-anneal before completion of the reaction. 
  • DNA degradation is often a by-product of the harsh bisulfite conversion reaction conditions. This can make working with smaller samples challenging.
  • Insufficient desulfonation of the reaction will leave behind residues that can inhibit DNA polymerases used in PCR. 
  • Recent evidence indicates that bisulfite conversion does not distinguish between 5-mC and 5-hmC 
    ​(Huang et al., 2010).
  • Bisulfite conversion lowers the overall complexity of the DNA sequence by essentially reducing the number of bases present from four to three. This can complicate primer design for downstream PCR-based interrogation or introduce challenges when attempting to uniquely map sequencing reads to a reference genome.

Bisulfite based DNA methylation applications

Based on its high utility, bisulfite conversion has become the basis for several variations and applications designed for higher throughput applications or investigation of broader, whole genome-scale regions.

Examples of bisulfite-based methods, which continue to be refined, include:

Targeted DNA methylation analysis

  • Bisulfite sequencing (BS-seq; Frommer et al., 1992) uses sequencing techniques to analyze bisulfite converted DNA samples. Initially, this was paired with standard Sanger sequencing, but now it most often refers to next generation sequencing protocols.
  • Methylation specific PCR (MSP; Herman et al., 1996) applies PCR primers specific to bisulfite converted DNA templates that are either methylated or unmethylated. The differential PCR amplification indicates if DNA methylation modifications are present. 
  • Pyrosequencing (Colella et al., 2003; Tost et al., 2003) is also known as sequencing by synthesis and can interrogate bisulfite converted DNA at a particular region of interest. The level of 5-mC is determined by comparing the ratio of C and T bases at an individual locus.
  • High resolution melting (HRM) analysis (Wojdacz and Dobrovic, 2007) was originally applied to SNP detection, but the process has also been adopted for DNA methylation. The real time PCR-based protocol measures melting temperatures of PCR amplicons. The shift in melting temperatures, which vary on C-T content, corresponds to the level of DNA methylation in the sample.
  • Methylation-sensitive single-nucleotide primer extension (MS-SnuPE; Gonzalgo ​and Jones PA, 1997) queries a CpG of interest by targeting bisulfite specific primers to the sequence immediately preceding a CpG. DNA polymerase terminating dideoxynucleotides allow the primer to extend a single base, which then can be quantitatively measured to determine C-T content, determining its DNA methylation status.

Genome-wide DNA methylation analysis

  • Whole genome bisulfite sequencing (WGBS; Lister et al., 2008) applies next-generation sequencing (NGS) techniques to bisulfite converted input samples. WGBS produces single-base resolution DNA methylation maps that span the entire genome of an organism. 
  • Reduced representation bisulfite sequencing (RRBS; Meissner et al., 2005) combines the single-base resolution of bisulfite , and the genome scale coverage of high throughput sequencing, with the use of methylation sensitive restriction enzymes to enrich samples for high CpG content. This approach effectively limits sequencing to only the regions of the highest interest, where DNA methylation exists.
  • DNA methylation microarrays (Adorjá​n et al., 2002) provide a genome-wide snapshot of DNA methylation through arrays of oligonucleotide probes targeting interesting CpG sites. Bisulfite converted DNA will hybridize to array features according to the DNA methylation status of each locus. The most widely adopted methylation arrays are the Infinium Methylation Assays, which use microarrays spotted with oligo-coated beads corresponding to a wide range of CpG sites.


The wide variety of bisulfite applications in play today exists because of the diverse needs of the research community. As no single method delivers the necessary results in every condition, those interested in studying DNA methylation in a variety of settings generally use multiple complementary approaches to obtain the data they seek.


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