degenerate primer desgining - degenerate primer desgining (Jun/18/2005 )

hello,
am trying to design degenerate primers for the amplification of a gene (from a plant)whose nucleotide sequence is not available. i've taken amino acid sequences from various sources and aligned them and taken the concerved amino acid sequence, in this case there is lot of degeneracy, so

should we consider the codon usage?

if it is to be consedered,how exactly should we do?

thanx
Ranadheer

You can consider codon usage, but usually people create primers which will match any codon. You'll find that the methionine residues provide good spots to anchor primers since it has a unique codon. I tried to make primers which were unique for as many bases as I could at the 3' end. For AAs which have six codons (e.g. Arg), you may have to make two sets of primers, unless you can avoid regions containing those amino acids. You might want to think about locations of introns, also, during this design process.

if it is to be consedered,how exactly should we do?

thanx
Ranadheer

hi group,
i have a doubt, related to degenerate primer desgining.
we r using the letter "Y" which represents C&T for desgining degenerate

primer, is that the "Y", single base that can bind to any of the 2 bases? or

is it used to mention the bases C&T for our convinience?

(not only "Y" other letters like "R" "W" "S" "K" "M" "D" "V" "H" "B")
thanking you
all
Ranadheer

The codes such as Y and R are codes for (C or T) and (G or A) respectively. When you specify a code such as this for a primer, the synthesizer mixes equal amounts of C and T (for example) at that base position. You end up with a mixture of primers, some of which have a C, and some of which have a T in that position. You should be trying to avoid this as much as possible, since it reduces the chances of all primers binding to your desired sequence.

Hi Mr.phage,

thankyou for reply. i am designing degenerate primers, in every third position there is ambiguity so i am using inosine.
so there are totally 6 inosines at every third position is it O.K?
and i am taking care so that there won't be inocine at the last base(i mean at the 3' position) does it actually matteres?
the sequense is like and has the same No of bases and the GC content is also around 50% is it O.K?
if you have any info about the degenerate primer designing information pls send me. thanking you
with regards
Ranadheer

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