PCR Cloning

Harry Witchel Harry.Witchel at Bristol.ac.Uk
Thu Jul 24 07:03:25 EST 1997


Dear Philip --
	You have some really nice amino acid sequence data, and it is 
fortuitous that most of the amino acids at the NH2 end are low 
degeneracy.  However, typically PCR requires two primers, which would 
suggest that you also want some protein sequence from the 3' end (which 
is not easy to get).  If you had the exact DNA sequence from the region 
that you have shown, then you could do one of several RACE protocols 
(with nested primers, or with a single Marathon primer).  HOwever, RACE 
is not best suited for degenerate primers.  You could design primers to 
KPDEED and antisense KGNFDV, and then you would have some non-degenerate 
sequence for the intervening region, but that region is too small to 
start a RACE project on its own.
	You also could screen a library using oligomers, but again, that 
technique is far better with non-degenerate primers.  Overall, my first 
choice would be to somehow get sequence from another part of the 
protein.  Also, it is worth noting that to do successful molecular 
biology, you should be willing to buy lots of primers, quite a few of 
which will not work -- if a reaction does not immediately work at 
all (given three temperature attempts eg 45C, 55C, & 65C) I typically 
have far more success buying new primers than optimizing the reaction.  
There are so many things to optimize (temp, cycle number, which enzyme, 
DMSO, Mg2+).  If you spend more than two weeks with a given primer set, 
you are wasting your time.  If financially a lab cannot afford to buy 
lots of primers, then the truth is that such a lab cannot afford to do 
molecular biology (which is one of the most expensive subfields of 
biology). 
	Barring that, here is a primer that you might try; it is based 
on the idea that exact matches for primers are only necessary at the 3' 
end of the primer, and that T can base pair inefficiently with G as well 
as A (and T is not so big that it interrupts the sequence when 
incorrect):

Lys Pro Asp Glu Glu Asp His Val
AAG CCT GAT GAG GAR GAY CAY GT

all the above choices for wobble correspond to the highest value for the 
codon usage table in humans (except for the T in aspartate).

For temperature values go to:
http://alces.med.umn.edu/rawtm.html

Also see the one page articles in Nucleic Acids Research:
"Minimal homology requirements for PCR primers" 1989 NAR 17:6749.
"Highly degenerate inosine containing primers specifically amplify rare 
cDNA using the polyemrase chain reaction." 1988 NAR 16:10932.

Best wishes, 
Harry


Dr. Philip M. Sass wrote:
> 
> I am trying to clone a membrane associated protein.  We have purified
> the protein (human) and managed to obtain 18 amino acids from the
> amino-terminus.  The sequence is KPDEEDHVLVLVKGNFDV.  Can anyone help us
> to design oligos for PCR cloning?  Any suggestions about PCR conditions?
> 
> thanks,
> 
> psass at uic.edu



More information about the Methods mailing list