PCR primers design

[John Nash]

In article <9305271216.AA20107 at ecc-s4.ecc.u-tokyo.ac.jp> s24303 at secc.ecc.u-tokyo.ac.jp (Nonaka Shigenori) writes:
>From: s24303 at secc.ecc.u-tokyo.ac.jp (Nonaka Shigenori)
>Subject: PCR primers design
>Date: 27 May 93 12:16:19 GMT


> Hi! We are trying to PCR-cloning by degenerate primers. What strategy
>is better to design primers deduced from AA-sequence?

>1.use Inosine for (A,T,C,G)mixture? or not?

I could never get this to work as well as using degenerate primers... and I 
tried hard.  Andre Hamel got better luck, I believe... my problem was that I
think I had too many inosines (5 or 6 in 20 - 25 mers).

>2.short primers with a smaller combinations of primers or 
>  longer ones with a bigger combinations?

This is a toughie... from experience, use the following guidelines:

a.  if you can, avoid "less-than-20-mers" with degeneracies... too many 
false primings.

b.  in my hands, up to 256 degeneracies are ok, if more degeneracies are in 
the oligo, split it into pools.  A quick Southern blot may tell you which 
alternative pool is worthwhile, especially if the pools are split along the 
lines of the codons for ser/leu/arg.

IMPORTANT:  If possible, make sure you have a nice run at the 3' end without 
degeneracies.  You'd be surprised at how many degenerate primers actually 
make the desired product, though, so if you can find a met and/or trp for 
the 3' end, at the expense of a few degeneracies 5' upstream, the non-
degenerate run at the end may be worth it.  I cloned the product of an 
amplification via degenerate primers, and compared the sequence with the 
wild type.... I found plenty of variation where the degeneracies in the 
primers was!

c.  when doing the pcr, I've found that longer anneals at higher 
temperatures give fewer false primings than playing around with lower anneal 
temps.  I used a 23-mer (192 degen) and an 18-mer (128 degen) to amplify a 
700 bp fragment, and a 55 deg anneal for 2 min was fine (I don't have a 
Turbo 9600, though).

>3.addition of restriction enzyme sites to both ends of primers
>  would be a drawback for the DNA amplifications?

Avoid anything which would lower the Tm further... adding ends is ok for 
exact match oligos, but for degenerate ones... I wonder if this would be 
beneficial.  I have never had problems cloning PCR products, and I don't use 
fancy cloning kits either.  What I do is add 1 ul of T4 DNA pol to the 
reaction directly after the PCR, leave it at 37 deg for 5-10 min, and kill 
the reaction, either with phenol or NaI (for "glass milk"- type cleanups).  
The two times I killed the reaction at 70 deg, I got nothing... does the T4 
pol exo activity go haywire before dying on the way from 37 to 70 deg??

>If they have been already discussed, please inform me.

>Thank you for cooperation.
>---
>Shigenori

Shameless Plug:  You could try my program PrimerGen (available from all good 
anonymous ftp sites) to generate oligo candidates.

Good luck.

cheers, John

John Nash                           | Email: Nash at biologysx.lan.nrc.ca.
Institute for Biological Sciences,  | National Research Council of Canada,
Cell Physiology Group.              | Ottawa, Ontario, Canada.
             *** Disclaimer:  All opinions are mine, not NRC's! ***