title: Assessment of the HPV DNA methylation status in cervical lesions.
abstract: 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.
authors.affiliation: Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of California Irvine, 114 Sprague Hall, Mail Code: 3905, Irvine, CA, 92697, USA.
authors.forename: Mina
authors.initials: M
authors.lastname: Kalantari
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Bernard
mesh_terms: D011232:Chemical Precipitation; D019175:DNA Methylation / Q000235:genetics*; D004273:DNA, Neoplasm / Q000302:isolation & purification; D005260:Female; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D027383:Papillomaviridae / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013447:Sulfites / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000821:virology*
chemical_list: D004273:DNA, Neoplasm; D013447:Sulfites; C042345:hydrogen sulfite