2 replies to this topic

[Raygoza]

Hi, our lab has a problem which studies the properties of DNA polymerases, and we are trying to identify an extremely rare mutation (deletion/insertion) that current sequencing technologies cannot detect with accuracy (i.e. the error is higher than the signal we're looking for) so i was wondering if we could use separation by capillary electrophoresis. Here's my rationale, capillary electrophoresis is used in sanger sequencing and thus is able to separate molecules that are 1 bp longer or shorter than each other, however, sanger sequencing is not an option for us give the huge cost that would involve. I'm looking to see if a service where capillary electrophoresis is detached from sanger sequencing and can work for us as an alternative for running a gel and for sequencing.

 

 

Thanks

Edited by Raygoza, 01 January 2015 - 02:57 AM.

[Trof]

PCR forum is not exactly the right one, I move it.

 

Yes, indeed capillary electrophoresis is used commonly for distinguising indels in the sequence, as small as one base, with fragmentation analysis protocols. Every sequencer should be able to do that, though it needs size standard, and sometimes different kind of polymer is better, some polymer types can be used for both.

The sequence detection systems are just universally running capillary electrophoresis, with any specified program, ask your sequencing company if the they can do fragmentation as well. Every "sequencer" is capable of doing it, but they may not offer it, but it's not a rarity.

 

What you need is a a set of primers amplifing your sequence, one of them fluorescent tagged, usualy FAM, compatible with the fragmentation standard they use (standards use a different dye). Use a proofreading polymerase. 

 

However, sensitivity of fragmentation analysis is a bit higher than Sanger sequencing (which is comonly around 10 %), but not when the mutation is extremely rare in the sequence.

 

I don't know what kind of tissue/cell type you are using that you get a very rare portion of a mutation, but in cancer tissues it may be so rare that is missed by fragmentation as well. But if you are searching for a known mutated sequence, designing allele-specific PCR is the most sensitive way of detection. Depends on how rare you expect it to be.

 

Fragmentation analysis is ideal solution for for detection of indels of different size, that are underrepresented in the DNA/cDNA.

[mdfenko]

do you have access to a capillary electrophoresis unit?

 

beckman-coulter has a kit for theirs for separating nucleic acids.

 

as trof said, sanger sequencing capillary electrophoresis units are also used for fragment analysis.