title: Impact of the DNA extraction method on 2-LTR DNA circle recovery from HIV-1 infected cells.
abstract: Detection of episomal 2-LTR DNA circles is used as a marker for the ongoing virus replication in patients infected with HIV-1, and efficient extraction of episomal DNA is critical for accurate estimation of the 2-LTR circles. The impact of different methods of DNA extraction on the recovery of 2-LTR circles was compared using mitochondrial DNA extracted as an internal control. The bacterial plasmid DNA isolation method extracted less than 10% of cellular DNA, 40% of mitochondrial DNA and 12-20% of the input 2-LTR DNA. The total DNA isolation method recovered about 70% of mitochondrial DNA and 45% of the input 2-LTR DNA. The total nucleic acid isolation method recovered 90% of mitochondrial DNA and 60% of the input 2-LTR DNA. Similar results were obtained when the DNA was extracted from HIV-1 infected cells. Plasmid DNA isolation could not distinguish between 12 and 25 copies of 2-LTR DNA per million cells, whereas the total nucleic acid isolation showed a consistent and statistically significant difference between 12 and 25 copies. In conclusion, the total nucleic acid isolation method is more efficient than the plasmid DNA isolation method in recovering mitochondrial DNA and 2-LTR DNA circles from HIV-1 infected cells.
authors.affiliation: Laboratory of Molecular Cell Biology, Frederick National Laboratory for Cancer Research, SAIC-Frederick Inc, Frederick, MD 21702, United States.
authors.forename: Yunden
authors.initials: Y
authors.lastname: Badralmaa
authors.forename: Ven
authors.initials: V
authors.lastname: Natarajan
mesh_terms: D004270:DNA, Circular / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification*; D015658:HIV Infections / Q000821:virology; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D010957:Plasmids / Q000302:isolation & purification; D013048:Specimen Handling / Q000379:methods*
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral
keywords: 2-LTR; Episome; HIV; Mitochondrial DNA; PCR; Retrovirus