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Ovided by the Light Cycler quantification software, version 3.5 (Roche Diagnostics). Two

by Burton Hannan (2019-04-25)


To quantify linear forms of HIV-1 DNA, a ligation reaction mixture was carried out by addition of linkers (final concentration: 30 nM) to DNA, in the presence of 10 units of PRIMA-1MetBiological Activity ligase from the Quick ligation kit (NEB), for 2 hours at room temperature in a final volume of 20 L, according to the manufacturer's instructions.

Linked DNA products were then purified with PCR switch charge purification kits (Life Technology) according to the manufacturer's instructions (to prevent inhibition of the PCR due to the mixture of ligation reaction) and eluted in 20 L and then submitted to real-time PCR.
In a first round of PCR, 1/10 of DNA was amplified in duplicate in a 20 l reaction mixture comprising 1 ?LightCycler FastStart DNA master Hybprobes (Roche), 4 mM MgCl2, 32 t and MS1 primers (300nM) forMunir et al. Retrovirology 2013, 10:87 website 14 ofquantification of the LTR5 (Additional file 1: Table S1B) or 32 t and 1LTR LA15 (5- CACACCTCAGGTA CCTTTAAGA-3) (300 nM) for LTR3.

To remain in the exponential phase allowing quantitative properties of the second PCR, 12 cycles are required for the first PCR. Decreasing the number of cycles for the first PCR leads a non-reproducibility in the samples quantifications (Additional file 1: tour malaysia giá rẻ Figure S6).
Increasing the number of cycles for the first PCR results in a false quantification because the exponential phase allowing quantitative properties of the second PCR is not respected (Additional file 1: Figure S6). 12 cycles are sufficient to ensure quantitative conditions for tour malaysia giá rẻ all linked-DNA dilutions for the second PCR.

The second PCR was performed on 1/100 of the first PCR-product in a mixture comprising 1 ?LightCycler FastStart DNA master Hybprobes, 4 mM MgCl2, 25 t and MS2 primers (300 nM) and hybridization probes MH FL and MH LC (200 nM) for quantification of the LTR5 (Additional file 1: Table S1B) or Tour singapore giá rẻ 25 t and 1LTRnested (5- TAK-243CAS GCTAATTCACTCCCAACGAAG-3) (300 nM) and hybridization probes LTR FL and LTR LC (200 nM) (Additional file 1: Table S1B) for LTR3.

Efficiency of the uDNAL quantification was determined by addition of the linker 11b to serial dilutions of the fragment from the digestion of pLIN-HIV-ScaI with ScaI and AatII. For pDNAL quantification, the efficiency of the procedure was determined by addition of the linker 11TAb (composed of oligonucleotides 25 t and 11TAb) (see Additional file 1: Table S1A) to serial dilutions of the fragment obtained after digestion of pLIN-HIV-NdeI with NdeI and AatII.

11GTb characterization as well as comparison between Southern blot and quantitative PCR were shown in Additional file 1: Figure S1).Method validationsensitivity of 10 copies for 200,000 cells) (Figure 1B). To assess the specificity of the quantification procedure, we tested the detection efficiency of pDNAL or uDNAL when using either 11TAb or 11b linker (Figure 1C).

We confirmed qualitative results from Pierson [22], i.e. 11TAb was not able to discriminate between pDNAL and uDNAL, while 11b led to detection of uDNAL only. From a quantitat.Ovided by the Light Cycler quantification software, version 3.5 (Roche Diagnostics). Two linkers: Linker 11b and 11TAb (or 11GTb in the virological context; see below) (see Additional file 1: Table S1A) were used for the quantification of uDNAL and pDNAL, respectively.