designing of probes - (Feb/12/2007 )
I need to design allele specific oligonucleotide probes for my project and i really dont know how to design....can anyone guide me in terms of any guidelines / reference / software / website that deals with it.....
thank you and have a great day
u need sequence of ur specific gene, and u need to determine ur purpose to design the probe
What kind of allele specific probes do you need? Are you just trying to make allele specific PCR and you want to design allele specific primers or...?
well my project involves studying various single nucleotide polymorphisms (SNPs) and for which i have to design probes (both wild and mutant type specific) which will hybridize with the pcr products. I have been able to standardize the pcr but i am having difficulties in designing the probes. There must be some guidelines for designing.
Any help will be greatly appreciated
I think the easiest way to detect SNPs is to use PCR primers with an intentional 3' mismatch or match at the mutation site. Typically these will not extend if there is a mismatch, at least with the correct annealing temperature.
If you want a hybridization probe, then you'd want to make an oligo with a relatively short sequence (12-15 bp I would think) with a central match / mismatch. The mismatch should ideally be a purine-purine mismatch (G matched with an A or vice-versa) because these are sterically harder to ignore. Many mutations, however, mutate C->T, and it's not possible to do this. You can use an oligo calculator to determine the melting temperature of the matched and mismatched probes -- you are trying to maximize the difference. There are probably also design rules for DNA hybridization arrays which could give you some clues -- they have exactly the same problem. Asking on that forum might be a good idea.
firstly thanks a lot....
why is it important to have a central match/mismatch ?
I had designed probes for a C -> T mutation but i observed that the wild type sample used to bind strongly even to the mutant probe..I tried different hybridization and washing temperature but all in vain, the nonspecific binding didnt reduce. The difference between the 2 probes was 2 degree. how big the difference can be?