Table 2.

Methods used to study human papillomavirus methylation

MethodDescriptionStrengthsLimitations
Nonbisulfite-based assays
Methylation-sensitive restriction digestionPair of restriction methylation sensitive and insensitive isochizomer endonucleases. Identification of methylation status by Southern blotting or PCR.Nonbisulfite method. Rapid and cost-effective, highly sensitive.Incomplete restriction digestion. Limited availability of restriction sites. Qualitative
Bisulfite-based assays
Quantitative methylation-specific PCRMethylation-specific probes allow for quantitative determination of methylation patterns and prevalence within a mixed pool of PCR products. Methylation can also be detected by methylation-specific primer design (methylation-specific PCR).High specificity and sensitivity has the ability to detect 1 copy of methylated DNA among several thousand. High throughput, quantitative.Detects methylation status of several CpGs simultaneously over a short genomic region.
Sequencing of individual clonesCloning bisulfite-treated PCR products into plasmid vectors and sequencing individual clones.Provides methylation information for single DNA molecules, allows for determination of variable methylation patterns.Slow, methylation status may be over or underrepresented as only a select few clones are chosen. Qualitative.
PyrosequencingSequencing PCR products for C/T polymorphisms, based on light-based detection of nucleotide incorporation.Rapid, high-throughput, for quantitative assessment of CpG site methylation, allows for assessments of DNA methylome.Provides an average value of percentage methylation.
EpiTYPERBase specific cleavage of transcribed RNA from PCR products yields distinct patterns for the methylated and non-methylated DNA, measured by mass spectrometry.High-throughput, quantitative assessment of CpG site methylation.Provides an average value of percentage methylation.
COBRAPCR amplification of bisulfite treated DNA followed by methylation dependent restriction digestion of methylation-specific sites.Provides semi-quantitative information about the methylation status at a specific region.Dependent on the availability of informative restriction sites.
Luminex xMapBead-based detection assay. Involves the hybridization and fluorescence detection of probes specific for the “C” of methylated or the “T” of unmethylated CpG sites coupled to microspheres.High-throughput, more sensitive than bisulfite sequencing.Detects only methylation frequencies.
Next-generation assays
IlluminaMultiplex, massively parallel next generation sequencing of PCR products for C/T polymorphisms.Analysis of the methylation status of individual HPV DNA molecules.Limited applicable statistical methods to handle the high-density, single CpG loci resolution data.
Pacific biosciencesDetects nucleotide binding to the polymerase in real time and is sensitive to base modifications, such as methylated CpG sites in the DNA template.High-throughput, no need for bisulfite modification of DNA.Not adapted to studies of HPV methylation, high error rate, and technically complex.