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
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
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:
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.
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?
> psass at uic.edu
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