Figure 1
From: Quantitative analysis of the time-course of viral DNA forms during the HIV-1 life cycle
Quantification of linear viral DNA. (A) Plasmids pLIN-HIV-ScaI and pLIN-HIV-NdeI are depicted. These plasmids were used as controls for the reaction specificity. DNAs mimicking uDNAL and pDNAL were obtained by ScaI/AatII and NdeI/AatII digestion of pLIN-HIV-ScaI and pLIN-HIV-NdeI, respectively. (B) Amplification of serial dilutions of the pLIN-HIV-ScaI plasmid. Parameters of the PCR (slope of the regression curve and intercept) using primers 25 t and MS2 are shown in the figure. (C) Amplifications resulting from the LM-PCR protocol, using the two linkers 11b or 11TAb in combination with DNA mimicking uDNAL or pDNAL. After ligation, all possible combinations were subjected to the LM-PCR protocol. The input was defined as the initial amount, determined by quantitative PCR. The output is the value obtained by quantification with the LM-PCR protocol. Efficiency is defined as the output:input ratio. The standard curves used for pDNAL and uDNAL quantifications in infected cells were obtained using 11TAb and 11b linkers, respectively (using serial dilutions of fragments obtained by NdeI/AatII and ScaI/AatII digestions of pLIN-HIV-NdeI and pLIN-HIV-ScaI, respectively). Right panel: PCR products from the various amplifications were loaded on agarose gel; M: Molecular weight marker.