ABSTRACT
We report a reliable method for PCR (polymerase chain reaction) amplification of genomic DNA from PET. This method uses DNA extraction with the QIAquick kit and amplification with AmpliTaq Gold. Amplification of up to 959 bp from PET was achieved with this combination which exceeds the current reported upper limit of 800 bp. In summary, the gradual activation of the AmpliTaq Gold during thermal cycling allows both for higher-fidelity and higher-throughput PCR amplification from PET. The use of the QIAquick kit for DNA purification of PET is sensitive, reproducible and suitable for management of a high number of samples.
Subject(s)
DNA, Neoplasm/analysis , Polymerase Chain Reaction/methods , Prostatic Neoplasms/genetics , 3-Oxo-5-alpha-Steroid 4-Dehydrogenase/genetics , Humans , Male , Mutation , Paraffin Embedding , Prostatic Neoplasms/chemistry , Reagent Kits, Diagnostic , Reproducibility of Results , Taq Polymerase/chemistryABSTRACT
We have investigated the antigenicity of the C- and N-terminal halves of pIX of human adenovirus types 2 and 3 (Ad2 and Ad3) as well as their orientations in virions. We found that only the C-terminal halves of Ad2 pIX and Ad3 pIX reacted in a subgenus-specific manner by enzyme-linked immunosorbent assay and immunoblot analysis. Based on immunoelectron microscopy experiments, pIX in viral capsids appears to be positioned such that the C-terminal part of pIX constitutes the surface domain whereas the N terminus of the protein makes up the internal domain in icosahedral Ad capsids.
Subject(s)
Adenoviruses, Human/chemistry , Antigens, Viral/analysis , Capsid Proteins , Capsid/analysis , Capsid/chemistry , Adenoviruses, Human/genetics , Adenoviruses, Human/ultrastructure , Animals , Antibodies, Viral/immunology , Antigens, Viral/genetics , Binding Sites , Capsid/genetics , Gene Expression , Humans , Neutralization Tests , Plasmids , Rabbits , VirionABSTRACT
Prostate cancer is a serious public health problem in many industrialized countries. Androgens appear to play a critical role in its etiology. Specifically, the active androgen in the prostate, dihydrotestosterone (DHT) which is synthesized by the enzyme steroid 5alpha-reductase from testosterone (T), acts as a mitogen. Hence androgen-deprivation is commonly used during prostate cancer therapy. Two isozymes for steroid 5alpha-reductase have been reported. The type II enzyme is prostate-specific and encoded by the SRD5A2 gene. We have investigated a polymorphic (TA)n dinucleotide repeat in the 3' UTR (untranslated region) of the SRD5A2 gene in 30 matched samples of constitutional ("germline") DNA from peripheral blood lymphocytes and microdissected, pure tumor DNA. We report here 8 LOH (loss of heterozygosity) events and 9 cases of microsatellite instability at this marker. Therefore, almost 57% of the samples examined showed evidence of somatic mutations at the 3' UTR of the SRD5A2 locus. Our data suggest that the SRD5A2 gene may be involved in prostate cancer progression and that this may have relevance for treatment of the disease.
Subject(s)
3-Oxo-5-alpha-Steroid 4-Dehydrogenase/genetics , Mutation , Prostatic Neoplasms/genetics , Aged , Dinucleotide Repeats , Disease Progression , Humans , Male , Middle AgedABSTRACT
Bacterially expressed recombinant protein IX (pIX) of human adenovirus serotype 2 (Ad2) and 3 (Ad3) was evaluated for use as a subgenus-specific antigen by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Patients sera positive by ELISA for the genus-specific adenovirus hexon antigen recognized the recombinant pIX of Ad2 and Ad3 in a subgenus-specific manner by both assays. Polyclonal rabbit serum raised against the recombinant Ad2pIX reacted strongly by indirect immunofluorescence assay, with Adl, 2 and 5 (subgenus C) but not with serotypes representing other subgenera. In a similar way, anti-Ad3pIX reacted with Ad3, 7, 11 and 14 (subgenus B), but not with serotypes representing other subgenera. A polymerase chain reaction showed that the complete pIX gene could be amplified in a subgenus specific fashion using primers specific for Ad3 (subgenus B), Ad2 (subgenus C), or Ad40/41 (subgenus F). The pIX gene from the available isolates of subgenus A, D and E was not amplified with these primers. The use of pIX-based serological assays is useful for subgenotyping as a primary screen of anti-Ad sera. It is much more rapid than the currently used neutralization assay or hemagglutination inhibition test. The application of anti-pIX sera by immunofluorescence and a pIX gene-based PCR are rapid methods which will improve subgenus identification of adenoviruses.
