Methods to Sequencing "Unknown" DNA - (Sep/15/2014 )

Hello,

I'm trying to characterize a naturally occurring deletion in an animal model.  The deletion spans about 10 Mb, which I've determined qualitatively through end-point PCR (Wildtype vs. Deletion Model).  This has been confirmed by FISH several years ago.  I've mapped the deletion breakpoints to regions that are highly homologous to each other, suggesting a non-homologous recombination (germline cell, X-chromosome) event may be responsible.

With the breakpoints mapped to areas within 1kb of the breakpoint, I've attempted to "span" the deletion with primers nested at each end.  This has failed.  I know this region has a high GC content, and I've tried additives such as betaine to enhance PCR - still no luck.

So, I've looked into PCR-based methods for sequencing the unknown.   Ligation-mediated PCR (LM-PCR and Inverse PCR are two that I've encounter.  I was unsuccessful with Inverse PCR, and have had limited success with LM-PCR.  LM-PCR seems to be limited to amplicons of less than ~1kb, and due to the high homology at the breakpoints, I believe my amplicons are larger than this bp size limitations.  (I must move further out with my primers to gain specificity, which makes my amplicons greater than 1kb).  I determined the relative limitation by using wildtype DNA as a control.

Does anyone else know of other techniques to sequencing the unknown?  I only have funding for low throughput (Sanger) sequencing.  I'm trying to avoid making a library, but will do it as a last resort.

Any input is greatly appreciated.

How did inverse PCR fail? Did you try several different enzymes? It has worked well for me in the past.

I'm unfamiliar with LM-PCR, but another technique which has worked for me is random digestion with an enzyme, followed by ligation of a known sequence adapter, followed by PCR from your known sequence and your adapter sequence. If you do this with more than one enzymes, you can often ping-pong between these ligation reactions, essentially primer walking the region, without doing anything more than making a new primer.

Hi Phage,

What you described in your second paragraph is essentially LM-PCR.    I can't get it to work at greater than 1kb amplicons. 

I never got the self-annealed DNA fragment of interest in inverse PCR; no exponential amplification was ever achieved.