To determine the solo-LTR region I first ran an electronic PCR on s240c3, s165c5 and s399c8 primers. I then aligned the PCR products to each other using a series BLAST searches, identifying a related sequence of ~968bp expected to be the same in each PCR product. Using BLAST I identified the start and end points of this sequence in relation to each of the PCR products of the 3 primers used in the experiment, allowing me to determine the direct repeats and thus pick out the Solo-LTR sequence and pre-integration site. From the size of the solo-LTR sequences, the size of the whole sequence and the identification of the direct repeat sequences I was able to work out the size of the preintegration site by taking the whole PCR product size and eliminating the size of one of the direct repeats and the entire solo-LTR sequence. The results are as follows:
968 = Solo-LTR size 584 = Pre-integration site size
967= Solo-LTR size 231 = Preintegration site size
968= Solo-LTR 295 = Preintegration site
Figure 1 Result of gel electrophoresis of my DNA sample
The product of the reaction of the A primer seems to have failed as no bands were produced apart from the terminal point of the migration which is too small to be considered as either a preintegration site or a retrovirus containing section, not only did my partner seem to have the same problem, most if not all of the submitted gels seem to have no bands for the A set of primers.
For the E primer sets, there are two strong bands. Around the 1018 MWM bands which approximately cor...
... middle of paper ...
...cluded here but ~15 were used in the search) apart from the last base on the direct repeat, so the sequence is unlikely to be totally irrelevant, it may represent a retro-element that the solo-LTR was derived from.
One method of identifying novel solo-LTRs within an individual would be to sequence their genome (pyrosequencing makes this somewhat viable) and align it to the current human genome sequence using NCBI blast, “jumps” may be noticed at certain point (i.e. a section which starts at base 260 in the human genome sequence but 960 in the individual sequence) the insert is likely to be due to a large retro element integration event, solo-LTRs can be easily confirmed by identifying direct repeats. You can then use knowledge of the flanking sequences to produce PCR primers to test for it in others.
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