Degenerate primers for PCR cloning

BOB RUTLEDGE BRUTLEDGE at AM.PNFI.FORESTRY.CA
Tue Mar 30 12:24:39 EST 1993


In article  <9303301002.AA15943 at net.bio.net> 
Lars.Snogerup at plantbio.lu.se (Lars Snogerup) writes

>Does anybody know anything about codon usage in plants 
>(Spinach oleracea). My reson for asking is that i am about to 
>order oligos for PCR-cloning, and i dont like the degeneracy 
>rate of the primers (1000-4000 times). I will add a cloning 
>handle to the 5' end of sense and antisense primers to be 
>able to clone the products into a vector, clone amp (any 
>hidden problems).

We have found that the primer degenerancy is important, in that 
primers with over 128 degeneracy do produce lower efficiency 
amplifications.  A simple solution that has been very effective for 
us is to include inosine at degenerate positions of 3 or 4 bases.  
This can dramatically reduce the primer complexity and avoids 
the use of codon usage (guesses) during primer design.  

We also include restriction sites at the 5' ends of our primers 
(Eco RI and Xba I) which work well... however, we are currently 
attempting to eliminate restriction digests of the PCR fragments 
by using T/A cloning.  Our early results have been encouraging. 
 

Finally, I would make two additional suggestions.  First is to use 
M13 as the cloning vector for PCR fragment subcloning.  This 
provides several important advantages that are central to 
allowing large numbers of PCR fragments to be collected and 
rapidly seqeunced, which has allowed us to character even very 
large gene families.  Second, keep the size of the amplified 
fragments <200 bp.  This allows the complete DNA sequence of 
each subclone to be easily  determined from a single 
sequencing run.  

Bob Rutledge
Petawawa National Forestry Institute
brutledge at pnfi.forestry.ca




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