Designing degenerate PCR primers-help

[indoubt]

i am looking for a tool where i can designe degenerate PCR primers.

by the way i have read about degenerate PCR, but find it hard to understand, so please explain these for me;

in standard PCR mixture we have a forward and reverse primer for our target, but how many primers do i need in a degenerate PCR mixture? besides how does the degenerate primers work? when we induce wobble in the primer, how come one of the specific one is chosen among the wobbles? or how does it actually work to make our pcr product? *extremely confused*



hope for ideas!

thanks a lot!

 

[iansmith]

I personnaly do not use tool design primers. I go with my own eyes. The best way to do it is to follow the following guidelines:

18-24 nucleotides (aim for 24)
45-55% GC (aim for 50%)
Melting Temperature ~ 60 °C (it usually more or less at 57)
Less than 4 nucleotides of self-complementation
No dimer formation at the 3’ end
G or C at the 3’ end (not A or T)
Avoid GGG or CCC at the 3’ end

For degenerate primers the idea is the that when you design the primer you have to decide to either put A, T, G, C or one the letter that represent more than one base pair. When the primer is synthesised, the machine will decided to to either base. For example, you design this primer ATGCRGTACC, R = A,G, so in your solution you will have a mix of ATGCAGTACC and ATGCGGTACC. The only is exception is the base I, which will be in all the primers since this base binds A, T, G, C. So in your primer stock you will have a mix of several differrent combination. The idea is that a some primers will have a good match and allow amplification.

 

[indoubt]

hi Ian!

so you mean that like standard PCR i have to design a forward and a reverse primer? and when design them i have to if necessary add the wobble in my primers and send them to a manufacturer. the manufacturer will make a combination of the different primers due to the wobble. and during the PCR only the primers which have the highest match with the target would bind to it?


hope for replies!
thanks!

 

[iansmith]

Everything you said is right

 

[indoubt]

many thanks!