wasowicz at moskit.art.olsztyn.pl
Fri Oct 24 01:03:34 EST 1997
"Meteorite" <vdmerwea at intekom.co.za> wrote:
>Could anybody give some pointers on how to go about designing degenerate
>primers? I have an amino acid sequence which revealed several conserved
>regions by comparison with related ones, but I want to know which ones I
>can use to design the primers from. Some are small (irrelevant?), while
>other regions are quite large (up to 10 amino acids). Also, I would like to
>know why some researchers include I (inosine) in their degenerate primers.
>What does I bind to, and what is its function in the primer?
>(Yours in nightmare...)
Basically inosine binds non-specifically to every nucleotide with the
same strength (at least as I know). Its presence doesn't affect the
melting point od the primer. If you have 25-mer and you have inserted
4 inosine residues you just treat your primer as 21-mer.
Some aminoacids are coded with one codon. Trp is coded by UGG. Then
you insert sequence TGG in your primer (or complementary - depends on
which primer you design - sense or antisense). Some aminoacids are
coded by four codons - Gly is coded by GGU, GGC, GGA and GGG. Then you
have two possibilities. You insert in your primer sequence GG (or
cmplementary) corresponding to first two Gs of Gly codon and then you
can insert mixed bases (A,C,T,G). Most commercial primer manufacturers
accept mixed bases for the price of one base. The problem with mixed
bases is that only one combination is the right one. With every mixed
base present in your primer the amount of the right primer decreases
correspondingly. If you have Gly codon and Pro codon processed like
this the amount of the right primer is only 1/16th of the total
amount. With every more mixed base it still decreases.
Some aminoacids are coded by more codons (Leu - 6 codons). You better
avoid such regions. You can exclude some codons when you look at the
codon usage table (should be available from EBI
(http://www.ebi.ac.uk). You can insert inosine at the third position
instead of (A,C,G,T). It will bind to every base nonspecifically, but
it allows to avoid problem with mixed bases. You just have to have
around 20 specific residues in your primer (If you have 4 inosines
make your primer 24-25-mer). Shorter primers may also work, longer may
be more specific but cost more.
Of course, nothing is as simple as it looks like. But what I have told
you may be a good starting point. I have been fairly successful with
inosine and 20-mers.
If you need some more advice, contact me.
Basically inosine binds to every nucleotide. If you need some more
advice, e-mail me.
Agricultural and Technical University
Faculty of Veterinary Medicine
Dept. of Animal Anatomy
10-718 Olsztyn-Kortowo, Poland
Phone: 4889-5233688, 4889-5233733
Email: wasowicz at moskit.art.olsztyn.pl
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