Titlebook: Human Papillomaviruses; Methods and Protocol Clare Davy,John Doorbar Book 2006 Humana Press 2006 DNA.PCR.Southern Blot.Virus.cell lines.gen

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Retrovirus-Mediated Gene Transfer to Analyze HPV Gene Regulation and Protein Functions in Organotypodology of retrovirus-mediated gene transfer, which can transduce the viral gene or genes into an entire population of PHKs. In this chapter, we describe the strategies and methods of retrovirus-mediated gene transfer into keratinocytes grown into organotypic cultures.

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Identification of HPV Variants,A→G mutation of the E6 ORF, using restriction fragment length polymorphism assays . In addition, we describe approaches for DNA sequencing and analysis. Such methods are likely to be of particular interest to those involved in epidemiological investigations of virus transmission and pathogenicity studies.

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Replication and Encapsidation of Papillomaviruses in ,,east provide a potentially high-efficiency means to produce large quantities of infectious virus in a short time frame. Fourth, assembly of HPV virus in yeast allows encapsidation of mutant genomes, since previous studies have shown that no viral ORF is required for replication of full-length HPV in yeast.

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Identification of New Papillomavirus Types,ere. The CP, FAP, and GP primers are used for PCR amplification, followed by cloning and sequencing of the amplicons. Sequence comparisons and the interpretation of DNA sequence identities are discussed. Details of defining a new papillomavirus type and of the recently approved taxonomic classification system for the . are given.

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Detection and Quantitation of HPV Gene Expression Using Real-Time PCR,sed to investigate the expression of HPV 16 early genes in HG or LG precancer are demonstrated. Detecting the expression of early HPV genes in conjunction with the Pap smear may improve the specificity of identifying LG precancers that are associated with high risk of progression.

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Analysis of p16INK4a and Integrated HPV Genomes as Progression Markers,ression of p16. is induced by the viral oncoprotein E7 and distinguishes dysplastic lesions from benign changes. Integration of human papillomavirus DNA into the host genome is mainly found in high-grade dysplastic lesions and invasive cancers, and points to an increased progression potential.