Hi folks,
I'm struggling to understand the PCR detection for correct MiMIC insertion (supplements Fig.2) in the Venken Paper "MiMIC a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes" (published 2011).
I am unable to find a way to relate the scheme to the PCR outcome.
Hopefully some of you know. Thank you.
Please see page 8 the manuscript (part of online methods).
Quote: For each RMCE event, four PCRs were performed: a first PCR
with primers Orientation-MiL-F and Tag-R, a second PCR with
primers Orientation-MiL-F and Tag-F, a third PCR with primers
Orientation-MiL-R and Tag-R, and a fourth PCR with primers
Orientation-MiL-R and Tag-F. As the transposon integrates one
or two orientations relative to the gene, only one in two RMCE
events is productive with respect to creating a gene trap or protein
trap, which is reflected in a positive PCR for reactions 1 and 4, or
2 and 3. A ‘1/4’ PCR pattern was always desired for a productive
RMCE event (for example, a gene or protein trap), when the gene
or transposon configuration is 1/1 or −1/−1. Conversely, a ‘2/3’
PCR pattern is diagnostic of a productive RMCE event, when the
gene or transposon configuration is 1/−1 or −1/1. The reverse
holds for unproductive RMCE events (Supplementary Fig. 2).
Thank you :)
" As the transposon integrates one
or two orientations relative to the gene.." I'd translate that like: the transposon can integrate either left to right or right to left. Is that correct?
" When the gene
or transposon configuration is 1/1 or −1/−1" I'm afraid we have a different way of notation in Germany. What does 1/1 mean?
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