Direct genomic sequencing - (Sep/29/2008 )
There is a disease in a population I work with that has a large deletion of unknown size. The problem is that I don't know the exact breakpoints of the deletion. I have high quality human DNA and I can't do PCR because I don't know what the downstream sequence is (because of the deletion). Is there any way to sequence (preferably with the BigDye Terminator chemistry) the DNA directly from the human genome (and if so, what is it)? I've devised the attached technique but I can't get it to work for the life of me... Any ideas what I could do?
How are you ligating your ends together? Normally you would add linkers to cut DNA, then clone them into a vector and go from there. There is a kit which might be helpful to you called the Genome Walker kit (from Clontech). Since you don't know how big your deletion is, you may end up sequencing a lot more than you want to since you have to start from a known region of DNA. It might be useful to do some further mapping using southern blots or FISH to help narrow down the region.
I'm ligating the fragments with T4 DNA Ligase (as does the Genome Walker kit). I found a research report stating that lower concentrations produced more circular DNA fragments and higher concentrations produced more concatamers, so I am doing a VERY dilute reaction (according to the research article, I can expect 95-100% circular DNA from this concentration).
The Genome Walker kit looks fairly complex, but it might actually work! I'll show it to my supervisor and see if we have enough money in the budget (we're a 100% non-profit lab and receive absolutely no government funding so money can be a little tight sometimes...) It somewhat reminds me of what I tried to do originally (before I came up with the protocol in the first post) which was use the Affymetrix 10K SNP array digestion and ligation protocol in conjunction with a primer specific to the last known "good" bases.
I do have another disease in the same population that I know the breakpoint for so I have been testing the protocol on one of these samples so that I'm not totally in the dark!
This is called inverse PCR, and works well in our lab (albeit for bacterial, not human genome sized organisms). You don't need to do the second restriction digest. The PCR will prime amplification of the uncut circular DNA (just as it does for a plasmid). Make sure your primers are oriented away from one another on the known sequence, and that there are no restriction sites between the primers and the known sequence ends.
I took some time and ordered primers that appear to work like the genome walker kit (I used a two base pair mismatch instead of an amine to stop PCR where it is not wanted and I used a different sequence for the "AP1" and the respective part of the probe-I actually used the sequence from the Affymetrix 10K SNP array primer). I digested the DNA and ligated the hybridized adaptor together as per the Affymetrix protocol, however when I ran the products on a gel-it didn't look good... Each set of primers made the same pattern, regardless of the enzyme being used (when there should have been at least a 2kb difference between some of the enzymes that I used with the same primer. In addition, a smear of fragments showed up in a reaction where I just the "GSP1" or the "AP1" primer ONLY. Clearly I'm doing something wrong or something isn't working. Any ideas as to what to do or how to fix it?