Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The sensitivity of human papilloma virus type 16 (HPV-16) DNA detection by DNA in situ hybridization using biotinylated probes (bio-DISH) was estimated by performing this technique on snap-frozen tissue sections of 10 cervical squamous cell carcinomas containing increasing amounts of HPV-16 as determined by Southern blot hybridization. A protocol using serial sections for bio-DISH and DNA extraction was used. The number of positively stained cells and the detection limit were strongly dependent on the treatment of the sections with proteinase K prior to hybridization. At low proteinase K concentration (0.1 micrograms/ml), the detection limit appeared to be 30-40 HPV-16 DNA copies per carcinoma cell, whereas morphology was preserved. A high proteinase K concentration (1-5 micrograms/ml) often resulted in an increase in the number of positively stained cells but also in a poor morphology. The detection limit was improved to at least 20 HPV-16 DNA copies per carcinoma cell.
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PMID:Sensitivity of in situ detection with biotinylated probes of human papilloma virus type 16 DNA in frozen tissue sections of squamous cell carcinomas of the cervix. 283 6

DNA from archival Papanicolaou stained smears was successfully amplified using the polymerase chain reaction (PCR) to see if it could be used for retrospective genome studies such as detection of the presence of human papilloma virus (HPV) and changes in p53 gene expression. DNA was isolated and purified by treatment with proteinase K, phenol/chloroform, and isoamyl alcohol. Segments of the human beta actin and TGF beta 1 gene were amplified by PCR. Of all stains used in the preparation of Papanicolaou smears, only eosin was detectable as a greenish band in ethidium bromide treated DNA gels under ultraviolet illumination.
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PMID:PCR amplification of DNA from stained cytological smears. 768 6

Accurate genotyping of a human papilloma virus (HPV) isolated from clinical specimens depends on molecular identification of the unique and exclusive nucleotide base sequence in the hypervariable region of a highly conserved segment of the HPV L1 gene. Among other options, a heminested (nested) polymerase chain reaction (PCR) technology using two consecutive PCR replications of the target DNA in tandem with three consensus general primers may be used to detect a minute quantity of HPV DNA in crude proteinase K digestate of cervicovaginal cells, and to prepare the template for genotyping by automated direct DNA sequencing. A short target sequence of 40-60 bases excised from the computer-generated electropherogram is sufficient for BLAST determination of all clinically relevant HPV genotypes, based on the database stored in the GenBank. This chapter discusses the principle and the essential technical elements in performing nested PCR DNA amplification for the detection of HPV from clinical specimens and short target sequence genotyping for HPV, using standard molecular biology laboratory equipment and commercially available reagents.
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PMID:Guidelines for the use of molecular tests for the detection and genotyping of human papilloma virus from clinical specimens. 2278 12