I tried to detect photosynthetic genes from an MDA-amplified genome of an uncultured green bacterial morphotype using degenerated primers by conventional PCR. I did not succeed and I only got unspecific PCR products. I suspect that if I try longer I will run out of sample and I do not have any more material. My question is: is it a better choice to clone such a MDA-amplified genome in order to screen more efficiently?. In this case: how is it possible to clone an MDA-amplified genome in order to sequence randomly selected clones looking for photosynthetic genes?. Thanks a lot in advance.
If you don't wish to express the gene in question, it may prove far more valuable to just sequence the entire genome. The miSeq platform from Illumina is probably your best bang for the buck.
At the very least you'd get some 15 million reads (double for paired end). UCDavis seq center is offering it for under US$ 1900 (plus library prep) . You'd also get all sorts of other interesting genes to go with the ones you are currently looking for. When you add the cost of cloning, screening and sanger sequencing, it definitely becomes worth considering the whole genome approach.
If having a clone/pcr product is a must, and you are reasonably sure your primers would work with the DNA you have, a touchdown PCR (to improve specificity) and an increased cycle number would usually help.
If you can, develop the protocol with a similar non-limiting sample (any other MDA amplified positive control would be fine, really), and when that works, move onto the real sample.
The last resort would be to do an old fashioned genomic bank, perhaps functionally screen it, and then sequence the function positive clones. but with the limited amount of DNA, this presents its own challenges.
Yes, to sequence the whole genome, i think, it is a better choice to screen functional genes you cared, as it is fast, cheap and less time cost. Based on the whole genome information, maybe you can found its regulation mechanism, which it is useful for genes to clone and express (this is what u will do next i think).
Many thanks for your answer. I guess that I do not have enough DNA to do whole genome sequencing in my two samples (less than 1 microgram). I wonder if a clone library would be the best choice. In that case, does anybody know how to clone MDA fragments?. I suspect that the MDA genome contains big DNA fragments that should be fragmented before cloning...
With 1 microgram of MDA amplified DNA, you should have plenty for Illumina Sequencing.
As a matter of fact, I just checked the illumina manual and the asked for amount is 1microgram for the shearing (using the covaris). I have personally used less, and on one occasion a little as 400 nanograms, on a chance that I'd get some sequences (I had MID's available for use on that lane): it worked like a charm: just over 1.2 million sequences for that sample. That was using HiSeq.
As for cloning, you'll have to bring the sizes to cloning range, so yes, fragmenting is in order. And you'll have to fill in incomplete strands and so forth, with clean-ups in between, which may reduce your DNA amount even further. a careful pilot will save you lots of grief int eh future :-)
If you decide to go with the cloning approach, I'd recommend you to use the AMPure beads from Beckman to do any clean up you may have: it is by far the most efficient recovery of DNA I've come across.
In our lab we usually would go for the whole genome sequencing and then do specific PCR for the genes of interest, to clone and (hopefully) express them for further characterization. This approach does work well in our hands (well, expression apart... that is hit or miss: you prob know how annoying that can be on Ecoli when it doesn't work right away...)
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