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.