Assessment of the HPV DNA methylation status in cervical lesions

The genomes of the human papillomaviruses HPV-16 and HPV-18 undergo increased CpG methylation during the progression of cervical neoplasia, possibly in response to increased recombination between viral and cellular DNA in high-grade lesions. This behavior makes HPV DNA methylation a useful biomarker of carcinogenic progression of HPV infections. The first step in detecting DNA methylation involves modification by bisulfite, which converts cytosine residues into uracil, but leaves 5-methylcytosine residues unaffected. A combination of this reaction with PCR and DNA sequencing permits to evaluate the methylation status of the sample DNA. This chapter describes the basic protocol to measure HPV-16 and HPV-18 CpG methylation by direct sequencing of the PCR products and discusses the value of modified strategies including DNA cloning, amplification with methylation-specific primers, and real-time PCR with TaqMan probes.