I was hoping to use the Surveyor assay from IDT to detect large (~600bp) deletions in a gene of interest. The wt amplicon should be ~1.2kb. My main questions are:
1) Is it even likely that the wt and mutated fragments will anneal?
2) If so, will the Surveyor nuclease recognize the sizable mismatch
and finally
3) If a mismatch is detected by the enzyme, where should I expect it to cut? At the start/end of the mismatch region? In the center? IDT doesn't offer much on this subject.
Any information would be greatly appreciated!
You should not need to use Surveyor to detect such a large deletion. If you amplify both wt and mutant fragments and run them on an agarose gel you will see two different sized bands.
Erin - I agree that, in general, that is the best course of action. To give some context, however: the amplified region was theoretically cut with the Cas9 enzyme at one or two target sites (near 5' and 3' ends of the amplicon, respectively), or at both sites. My results do not indicate a single cut at either site, but rather seem to suggest that the ROI was cut somewhere in between the two targets. I'm wondering if this could be due to a collapse and/or indel formation between the two sites. I apologize for not being more clear.
Oh ok, I guess I see your problem, thanks Rogan. It still seems to me that if you have a large indel you can detect it just by PCR. If the indel is small Surveyor will detect, but I think the results may be messy if the indel is over 20bp or so. Others may have more experience with this...
That was what I assumed, as well. In any case, your input is very much appreciated!
- Badges
- Science topic