Use of a rapid and simple method to extract proviral DNA in the identification of HIV-1 by PCR.
DNA extraction is a critical step in PCR analysis and is closely related to its sensitivity. Traditional methods, based on phenol-chloroform extraction, require more time and the use of toxic reagents. GeneReleaser (Bio Ventures Inc.) is a commercial product which releases DNA from whole blood, cell cultures, bacterial colonies and the like. Cells lysis and DNA extraction are accomplished directly in the amplification tube on a thermocycler. We used GeneReleaser in the identification of HIV-1 proviral DNA by PCR on whole blood samples. All samples arrived at our laboratory for HIV-1 detection were treated with two different procedures. The classical one was based on the lysis of separated lymphocytes by proteinase K, while the other consisted in DNA extraction by GeneReleaser from 5 microliters of whole blood in sodium citrate. All samples were amplified for HIV-1 GAG region; to prevent carry-over contamination Uracil N-glycosylase (UNG) sterilization was performed. Amplified sequences were revealed using the DEIA commercial system (Sorin Biomedica, Italy). To verify the suitability both of cell lysates and GeneReleaser DNA-extracted samples for PCR, we amplified a specific sequence of HLA-DQ-alpha gene. Initial data indicate that this new method might reduce the performance time of PCR (DNA extraction time was around 15 minutes) and improve PCR sensitivity.
Tagliaferro
L
L
Dept. Virology, Laboratory Pignatelli, Lecce, Italy.
Corbelli
M
M
Maietta
G
G
Pellegrino
V
V
Pignatelli
P
P
D000328:Adult; D001773:Blood Cells / Q000821:virology; D004247:DNA / Q000302:isolation & purification; D045647:DNA Glycosylases; D017931:DNA Primers; D004260:DNA Repair; D019286:Endopeptidase K; D005260:Female; D015750:Genes, gag; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis*; D015497:HIV-1 / Q000302:isolation & purification*; D006683:HLA-DQ Antigens / Q000235:genetics; D006801:Humans; D007231:Infant, Newborn; D008214:Lymphocytes / Q000821:virology; D008297:Male; D009699:N-Glycosyl Hydrolases / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000302:isolation & purification*; D012697:Serine Endopeptidases / Q000737:chemistry; D051981:Uracil-DNA Glycosidase
D017931:DNA Primers; D006683:HLA-DQ Antigens; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D012697:Serine Endopeptidases; D019286:Endopeptidase K