Suggestions for primer design - How many bonds is "too many" to avoid dimers and hairpins? (Oct/31/2009 )
I am a newcomer to molecular biology and currently undertaking my first project. I am but a mere ecologist, but I am (trying) to use molecular methods to investigate the diets of wild bats, by amplifying insect DNA from bat feces. I using mtDNA (COI region) for this study.
I am trying to design a number of species-specific primers to find out if these bats are eating any of the major agricultural pests in my study area. Because I am working with samples which could have nearly any insect in them, my primers *must* target areas that are unlikely to amplify DNA from non-target species. This limits me somewhat in the regions where I can situate my primers.
I have used PrimerBLAST (NCBI) to identify a number of potential primers for my target species. However, all of these primers have potential problems with forming dimers and/or hairpins. However, for some these seem to be minor issues -- e.g., dimers with only two or three strong bonds, and not at the 3' end.
While I can find plenty of info on how you don't *want* dimers, I'm having less luck trying to determine what level of dimer potential is "acceptable" -- is there some sort of criterion or cut off at which you can say "yes, you will have problems with dimers" if you have X number of bonds?
I don't think you'll find definitive guidance -- the proof is in the function. Primers are sufficiently cheap that it is worthwhile to simply try them. A 3' mismatch will almost certainly prevent an extension, and 3 bp matches don't sound as if they would do anything.
i dont think a 2-3 base pair wiould make a difference..you can easily play around with the annealing temperatures during your PCR to avoid thses.....good luck to you!!!!!!!