Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Methods to Sequencing "Unknown" DNA


  • Please log in to reply
2 replies to this topic

#1 djvan

djvan

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 30 posts
0
Neutral

Posted 15 September 2014 - 09:25 AM

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.

 

 

 

 

 

 

 



#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,484 posts
251
Excellent

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

djvan

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 30 posts
0
Neutral

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.

 

 






Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.