PCR gene splicing: help!

pskaytes at INTNET.UPJ.COM pskaytes at INTNET.UPJ.COM
Mon Oct 10 09:49:03 EST 1994


Kevin:

I've recently run into the same problem, and think I've found the solution.  
Remember, many of the polymerases add an additional nuclteotide to the 3'
end of the products.  When the initial products are annealed for the second
round of PCR, this may (3 out of 4) produce a mismatch, and worse yet, at
the 3' end of one of the elongation points, just where it can do the most
harm.  There are two possible solutions:  For the second round, you can use
a polymerase with proofreading ability, that is, 3'->5' exo activity, that
will repair the mismatch, and then extend.  Alternatively, you can plan the
overlap so that the added base is not mismatched.  For that, I recommend you 
take a look at Gengxi Hu's paper in DNA and Cell 
Biol.  vol 12, 763-770  to predict the nucleotide which will be added.  Hope
this helps.
Paul Kaytes
pskaytes at intnet.upj.com



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