Degenerate Primers

John Nash nash at nrcbsa.bio.nrc.ca
Tue Sep 21 20:46:29 EST 1993


In article <9309221821.AA13494 at umailsrv0.UMD.EDU> William_D_WARREN at UMAIL.UMD.EDU (ww40) writes:
>From: William_D_WARREN at UMAIL.UMD.EDU (ww40)
>Subject: Re: Degenerate Primers
>Date: 22 Sep 93 18:20:00 GMT

>>  The Molecular Cloning Manual (Sambrook, Fritsch, & Maniatis) discusses
>>the rational of synthesizing degenerate PCR primers by hand, but I would
>>like to find out if there are any computer programs available that will
>>generate degenerate primers if given a set of known sequences (I have
>>sequences from 10 or so other species).   I looked into Bill Engles Amplify
>>program, but it doesn't seem to handle degenerate primers.  Will GCC allow
>>me to do this? 
>>
>Karl,

>TO my knowledge there are not any programs currently available that will, if
>fed a multiple sequence alignment, will spit out optimized degenerate PCR
>primers. Although I have no doubt that such a program could be written, the
>task is a complex one and it would be far from trivial (if it were a trivial
>task then you'd not be asking for a program to do it for you).

I have occasionally hand fed peptide fragments gleaned from multiple 
protein alignments to my program PrimerGen - available as PGEN.EXE from many 
good ftp sites and files servers, and also hidden in a corner of the recent 
CABIOS ;-)  PrimerGen is designed to fish out nice degenerate primers from 
peptide strings.  Alas, it's a PC application, as I am still in the middle 
of the UNIX port... I'm still learning UNIX.

It works fairly well - a lot easier than trying to fish such primers from 
multiple alignments of DNA (which make me cross-eyed)... I have generated a 
number of oligo probes this way.

>I have designed many pairs of matched degenerate primers that have worked
>successfully and I have done them all by hand. I personally wouldn't trust a
>black box program to do the task for me.

It depends on how well the "black box constructor" explains things.  
There is always going to have to be some sort of decision/call on the part 
of the researcher as to which primer sequences will ultimately be fed to 
the synthesizer, but a computer program can give one a nice range of 
choices rather than the whole set of alignments.  I'm toying with 
eventually writing such a program, but I am reluctant to battle with the 
different multiple alignment formats out there (I'll probably settle on GCG 
and FASTA since these are my favourite program suites).  Also, I need to 
have a personal research reason to do this........... but if I do, the net 
will be the first to know.

>If you would like some basic DO's and DON'T feel free to Email me.

Actually, if you post, I'll append some of my 
blunders^H^H^H^H^H^H^H^Hexperiences to Bill's tips.

John Nash							 Email: nash at nrcbsa.bio.nrc.ca

Institute for Biological Sciences,	National Research Council of Canada.
	*** Disclaimer:  All opinions are mine, not NRC's! ***



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