Tated K65R cDNA were mixed and sequenced; peak heights wereTated K65R cDNA were mixed and

Tated K65R cDNA were mixed and sequenced; peak heights were
Tated K65R cDNA were mixed and sequenced; peak heights were measured for both nucleotides and UNC0642 cost percentage PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28914615 reversion was calculated according to our previously published protocols [14]. To confirm the ratios of peak heights observed, we performed population cloning in Topo TA cloning vector PCRR2.1 (Carlsbad, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28827318 CA) by cloning RT PCR products and sequencing 20 clones at each time point.Chunduri et al. Virology Journal 2011, 8:33 http://www.virologyj.com/content/8/1/Page 4 ofIn vitro RT processivity assaySince various viral (nucleocapsid proteins, integrase) and host factors (p53 and cellular topoisomerase) have been shown to interact with HIV-1 RT [16-23], we compared virion-associated RTs of mutant and wild type viruses in all of our assays. RT processivity assays were performed as described elsewhere [13,24,25]. Briefly, stock viruses supernatants containing 1500 to 3000 ng equivalent of antigen p24 were centrifuged at 16,000 rpm for 2 h at 4 . RT was dislodged from the pelleted virions by the treatment of 50 l of 0.5 NP40. The RT activity was determined using homopolymer template/primer [poly rA-oligo d(T)] and a-32P dTTP according to published protocols [12,15,25]. Different amount (2 l, 4 l, 6 l) of RT lysates were incubated with 1 g/ml of poly (rA) and 0.16 g/ml of oligonucleotide (dT) in the presence of an assay mixture containing 60 mM Tris (pH 7.8), 75 mM KCl, 5 mM MgCl 2 , 0.1 NP40, 1 mM EDTA, and 4 mM DTT at 37 for 30 min in the absence of radiolabeled dTTP. After the formation of Template-primer-enzyme complex, cDNA synthesis was initiated by the addition of 50 Ci of [a-32P] dTTP/ml and 50-fold excess of trap [poly (rC)-oligo (dG)]. The reactions were terminated after 180 min by placing the tubes in ice slurry and addition of the equal volume of buffered phenol. cDNA products were extracted once with phenol:chloroform (25:24) followed by one extraction with chloroform only. In order to normalize the volume of extracted cDNA, equivalent amount of top layer (DNA) was collected after centrifuging the mixture of phenol and DNA solution. The cDNA was precipitated with 2.5 volumes of absolute alcohol in the presence of 2.5 M ammonium acetate. After desalting the precipitated DNA with 70 alcohol, the pellet was vaccume-dried and suspended in 8 l of sterilized water. Half of the DNA was mixed with formamide-dye mixture and heated at 95 for two minutes in a water bath. The purified products were run on 6 polyacrylamide sequencing gel electrophoresis at 30 W for 2 h. The wet gels were exposed to autoradiography for 30 min to 2 hr. To determine relative density of bands in the gel, we scanned group of bands using Bio Image Intelligent Quantifier ?software (Bio Image Systems, Inc, Jackson, MI).Statistical analysisobserved and expected peak heights for two nucleotides at the same locus [14]. Statistical analysis was conducted to determine the differences in processivity between WT and mutant viruses or among mutant viruses K65R +L74V and K65R+L74I during a single processivity cycle. This analysis was designed to test the hypothesis that for wild type and mutant RTs, cDNA density decreases at the same rate as DNA band number increases. Three to five independent processivity assays were performed for each RT and statistical values that include mean, median, standard deviation and maximum and minimum were obtained. A paired analysis with ttest was performed to compare the density of cDNA products generated by various RTs and p 0.05 was co.