Take the 2-minute tour ×
Biology Stack Exchange is a question and answer site for biology researchers, academics, and students. It's 100% free, no registration required.

Let's say I have two fragments of bacterial DNA (~50kb in length each) that overlap on the ends by roughly ~10kb; the overlap sequences are unknown. I would like to assemble these into longer fragments (in vitro) in some way, but the methods I've found so far rely on 24-40bp known overlaps (e.g., the isothermal assembly process in DG Gibson et al., Nature Communications 2009).

Anyone have any idea? I'm kind of new to this molecular biology stuff, so if this is an open problem, I apologize. I'm mainly just making sure I'm not overlooking something obvious. For all I know, there could be a standard protocol for this. Thanks in advance!

share|improve this question
1  
As far as I can see, typical overlaps are between 20 and 100 bases. These extreme long overlaps may generate some "funny" results which may result in a DNA that you don't want. What do you know about the bacterial DNA you are working with? Can you sequence the ends to find out how big the overlap is? –  Chris Jul 8 at 16:10
    
Thanks for your comment! My problem is essentially that I need fairly long DNA fragments from cells, but the extraction process tends to break them apart more than I would like. I'm just wondering if there exists some method to re-assemble them after extraction without sequencing. The overlap may be anywhere from nothing to several kilo-bases. –  Chuck N Jul 8 at 16:36
    
Can you use a different extraction method? Look into methods to prepare BACs from bacteria, they are several hundret kB in size and need to be prepared intact in order to use it further. –  Chris Jul 8 at 16:50
    
Good point. I currently get the DNA already-fragmented from a collaborator and could request a different method for future purposes. I thought if there were some way to easily assemble the ones I already have then it would be worth a shot, and if there wasn't a method already, then it would at least be interesting to think about :) –  Chuck N Jul 8 at 16:57

Your Answer

 
discard

By posting your answer, you agree to the privacy policy and terms of service.

Browse other questions tagged or ask your own question.