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Methods to Sequencing "Unknown" DNA

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#1 djvan



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Posted 15 September 2014 - 09:25 AM



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.








#2 phage434



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Posted 15 September 2014 - 01:28 PM

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.

#3 djvan



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Posted 16 September 2014 - 06:03 AM

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.



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