Journal: Journal of Virology
Article Title: Specific Cleavages by RNase H Facilitate Initiation of Plus-Strand RNA Synthesis by Moloney Murine Leukemia Virus
Figure Lengend Snippet: Extension from PPT62 at a nick followed by the M-MuLV RNA. 5′ end-labeled PPT62 without a downstream oligonucleotide (none) (lanes 1, 4, 7, 10, 13, and 16 to 19) or with downstream MLVnick (lanes 2, 5, 8, 11, 14, and 20 to 23) or MLVnickD (lanes 3, 6, 9, 12, 15, and 24 to 27) was annealed to template 2. Extensions were carried out with T7 DNA polymerase (T7; lanes 4 to 6), T4 DNA polymerase (T4; lanes 7 to 9), RTΔH (lanes 10 to 12), wild-type reverse transcriptase (wt RT; lanes 13 to 15) or H − RT (lanes 17 to 19, 21 to 23, and 25 to 27) for the indicated times. Substrates incubated without enzyme are shown in lanes 1 to 3, 16, 20, and 24. The products were analyzed in a 20% sequencing gel and visualized using a PhosphorImager. A schematic of the nicked substrate II tested is shown at top, and the positions of unextended PPT62 and the full-length extension product are indicated on the right.
Article Snippet: RNA oligonucleotides were obtained from Oligos Etc., DNA oligonucleotides were purchased from Invitrogen, and all oligonucleotides were gel purified prior to use.
Techniques: Labeling, Incubation, Sequencing