5' RACE-Tail cDNA with dC, or dT?

Reese Bolinger anmab2 at orion.alaska.edu
Thu Oct 5 00:50:28 EST 1995


Dear GLarson:

I have some limited experience with 5' RACE, and when I performed it, I 
tailed my first strand cDNA with dATP.  Perhaps the concern here is two 
fold...1).  the higher Tm of a poly dC tail relative to a poly dA tail.  
2). the poly dA primer/adaptor can be utilized for both 5' and 3' RACE

Regarding the use of your degenerate primer for the initial PCR 
reaction...when I've performed this, I've always primed with one 
gene-specific primer during first-strand synthesis and carried out the 
PCR with a second gene-specific oligo.  In theory, you should be able to 
carry out your PCR with the same oligo(s) you primed your mRNA with.  In 
fact, in CPMB's protocol for 5'RACE, they have you do two seperate PCR 
amplifications: first with the oligo used to prime the mRNA and then re 
amplify with a second gene specific oligo.

The only problem I can forsee with doing the PCR with the same oligo 
used to prime the mRNA is one of specificity.  If your primer is 
degenerate, you will get more "false" priming to begin with, which the 
use of a second gene-specific oligo would help to minimize.  So if you 
only use the single set of degenerate primers, you will probably want to 
subclone and do colony screening on your transformants prior to 
sequencing to take care of that problem.

Hope this helps.....

                                           Reese





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