Here's another technical problem for the new plans. We want to reisolate and sequence DNA that competent cells have taken up into their cytoplasm. We'd start with a large number of competent cells carrying a rec-1 mutation that prevents them from recombining DNA with their chromosome, and incubate them with genetically marked DNA fragments. We expect the DNA to be taken up (it would contain one or more USSs) across the outer membrane and then translocated into the cytoplasm. The problems arise because, although the process of translocation starts at a free end of double-stranded DNA, one of the strands is degraded as the DNA is translocated. This means that the DNA in the cytoplasm will be single-stranded.
We can treat the cells with DNaseI to destroy DNA that's left outside the cells or on their surfaces, and then wash them thoroughly t remove the DNase I so it doesn't act on the DNA inside the cells. We can then lyse the cells with SDS in the presence of EDTA, being very gentle so to not break the long chromosome.
Getting rid of chromosomal DNA is very important, as there will be at least 10 times as much of it as the incoming DNA we want to reisolate. If we start with input DNA that is of a uniform and relatively short length, we will be able to use size fractionation to get rid of most of the chromosomal DNA. And we probably can further enrich for the input DNA by fractionating on a column that selects for single-stranded DNA.
One solution would be to affix a sequence tag to the ends of the input fragments, and then use a primer for this tag to PCR-amplify the input DNA. Unfortunately, the leading (3') end of the surviving incoming strand is also thought to be degraded, so the tag would probably be lost. As this is the end the PCR would start from, the PCR then wouldn't work.
We don't want to tamper with the structure of the incoming DNA, as this is likely to interfere with normal uptake in some way we can't control for. And we don't want to use recipients with nuclease knockout mutations, partly because we don't even know which nucleases are responsible and partly because we don't want to pervert the normal uptake/processing events.
One possibility is to use a combination of tagging and random priming, with the lack of tags on the 3' ends compensated by the random primers. Maybe we could test this, using radioactively labelled input DNA with tags. If the method is working, most of the input radioactivity in the reisolated DNA would be converted to double-stranded. Or we could test it using DNA from another species, and sequencing a small fraction of the PCR products to see if they were indeed from the input genome rather than from the recipient.
Because we're really only interested in the relative proportions of different input sequences in our reisolated DNA, we can tolerate a modest fraction being from the recipient genome. But we don't want to waste our expensive sequencing dollars on DNA that's mostly from the recipient.
Friday Fabulous Flower - Palm flowers
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