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Essay On Human Papillomavirus

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Human papillomavirus are miniscule double stranded DNA viruses which target cutaneous and mucosal epithelial cells, inducing lesions which may lead to hyper proliferation and tumorigenesis. There are over 100 identified strains of HPV which are classified into two types; Low-risk types which may lead to symptoms such as benign genital warts in contrast to high risk types which may lead to aggressive tumorigenesis and metastasis.

The genomes of all HPV strains contain nearly eight open reading frames (ORF). Each when transcribes produces a polycistronic mRNA( meaning that the mRNA can code for a multitude of different proteins). The regulatory sequences that are needed for the regulation of transcription of these polycistronic mRNAs …show more content…

Genther et al, 2003). E5 also appears to encode a membrane protein with a weak transforming activity (Leptak et al., 1991). E6 and E7 however are the main transformants involved which may lead to mutations that cause tumorigenesis. Thus the first step of this process is to obtain a high concentration of wild type HPV31 viral plasmid in order to be able to adequately re-engineer for the treatment.

A sample HPV31 will be bought an injected into several cultures of HeLa cells grown in DMEM+FBS, we will also have a control plate where no HPV31 will be injected. This will ensure that there is no contamination in our plates. Some of the live HPV31 will be set aside to extract their viral plasmid and sequence them either using new generation techniques or Sanger sequencing. The HeLa cultures infected with HPV and the control plate will be allowed to grow for two weeks to allow the proliferation of the HPV viral plasmid.

Once incubation is complete the viral DNA plasmid will be extracted from the cultures using a mini-prep protocol as follows. Cells will be washed twice with PBS and lysed in lysis solution (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM EDTA, and 0.2% SDS) for total genomic DNA extraction. The cell lysates will treated overnight with proteinase K (0.2 μg/ml), extracted with phenol-chloroform and precipitated with 2 volumes of 96% ethanol. DNA pellets will be dissolved in TE buffer containing 20 μg/ml RNase A and incubated for 1 hour at 37°C. The DNA will

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