francis at borduas.nlm.nih.gov
Sat Apr 9 16:49:06 EST 1994
Here is a section from Paul Hengen's FAQ dealing precily with the
question of oligo/PCR and software ...
the full document can be retrieved from a bionet archive site near you
.. I picked this copy up from the Indiana gopher server:
| B.F. Francis Ouellette
| francis at ncbi.nlm.nih.gov
Subject: FAQlist for bionet.molbio.methds-reagnts
Sender: Paul N. Hengen
Date: Wed, 23 Mar 1994 18:16:25 GMT
* Frequently Asked Question (FAQ) list *
* for bionet.molbio.methds-reagnts *
* 23 March 1994 *
16. What kinds of programs are available for designing PCR primers?
There are four programs which deal with pcr primer construction which one
can obtain by anonymous FTP and one which will be mailed to you by the author.
The programs are:
1. Pgen - for DOS only
PrimerGen searches strings of amino acid residues in order to reverse-translate
oligonucletide primers of a desired range of lengths and maximum number of
PrimerGen only works on IBM-PC(TM), XT, AT, PS/2 and compatibles with
EGA or VGA graphics adaptors. It will not work on computers with CGA or
Hercules(TM) graphics cards. A hard drive is NOT required and PrimerGen will
fit on a 360K 5.25" floppy disk.
PrimerGen contains a sequence editor where amino acid residues are entered.
The amino acid sequence must be ONE fragment and cannot be longer than 70
residues. The sequence must be in the ONE LETTER CODE and cannot contain
any UNKNOWNS. After the desired amino acid sequence has been entered have the
option of saving the sequence to a disk. PrimerGen will also accept and
re-edit previously saved sequence files, and also contains a codon preference
table editor. You can get this program by anonymous FTP at
ftp.bio.indiana.edu. It is found in the molbio/ibmpc directory.
2. Primer - Stanford - Sun Sparcstations only
The program 'primer' is written by Don Faulkner. It helps to find potential
mispriming sites (primer sequences should be designed before running the
program!). The program gives higher weights to matches at the 3'
end of the primer, linearly decreasing them towards the 5' end (the default
is weight=10 for 3' nucleotide decreasing to 1 at nucleotide # 8 from the 3'
end). The program can be used when amplifying *long* fragments from a known
sequence. The program is written in "C" and runs on Sun workstation (Unix).
You can get the program by contacting James Mullins at Stanford University.
[e-mail: jmullins.stanford.edu phone: (415) 723-0668]
3. Primer - Whitehead -Unix, Vms (and DOS and Mac if you can compile it)
PRIMER is a computer program for automatically selecting PCR primers
written by Steve Lincoln, Mark Daly, and Eric Lander.
PRIMER will run on just about anything which supports a standard C
- IBM PC and compatibles. A PC/AT (286) class machine or better with 640K of
- Apple Macintosh computers. A Mac II series or SE/30 with a math coprocessor
- Most Unix workstations including Sun SPARCStation and DEC DECStation systems
- DEC VAX/VMS computers under VMS Version 5 with VAX C.
PRIMER is available from a number of sources:
- You can get PRIMER from another user, provided you strictly follow the terms
- You can download the most recent version off the Internet via anonymous FTP
- You can send a request to:
PRIMER c/o The Lander Lab,
9 Cambridge Center, Cambridge, MA 02142.
E-mail to "primer at genome.wi.edu" (Internet), or FAX to 617-258-6505
You can also get PRIMER by anonymous FTP from genome.wi.edu in the
4. Amplify - MAC only
This software is for use in designing, analyzing, and simulating
experiments involving the polymerase chain reaction (PCR). PCR is a
technique used by molecular biologists to amplify highly selected
segments of DNA.
You can obtain a copy of Amplify from
sumex-aim.stanford.edu via anonymous FTP. Look for the file:
The author, Bill Engels, can be reached at:
Phone: (608) 263-2213
lab: (608) 262-5578
Fax: (608) 262-2976
E-mail: wrengels at facstaff.wisc.edu
5. OSP - Unix, X-windows, Vms, DOS, Mac - by snail-mail only.
OSP is available for free, but the university lawyers require that you
sign a licensing agreement. The legal document is not for the
paperwork faint-at-heart. It is quite long and daunting. If you're
waiting for the legal stuff before starting your experiment, you
may be better off working out a primer by hand.
Here is the abstract from the paper describing OSP, which appeared in
PCR Methods and Applications 1, 124-128 (1991), along with information
on how to get the program.
OSP (Oligonucleotide Selection Program) selects oligonucleotide primers for DNA
sequencing and the polymerase chain reaction (PCR). The user can specify (or
use default) constraints for primer and amplified product lengths, %(G+C),
(absolute or relative) melting temperatures, and primer 3' nucleotides. To help
minimize non-specific priming and primer secondary structure, OSP screens
candidate primer sequences, using user-specifiable cutoffs, against potential
base pairing with a variety of sequences present in the reaction, including the
primer itself, the other primer (for PCR), the amplified product, and any other
sequences desired (e.g., repetitive element sequences in genomic templates,
vector sequence in cloned templates, or other primer pair sequences in
multiplexed PCR reactions). Base pairing involving the primer 3' end is
considered separately from base pairing involving internal sequences. Primers
meeting all constraints are ranked by a ``combined score'', a user-definable
weighted sum of any of the above parameters.
OSP is being routinely and extensively used to select sequencing primers for
the C. elegans genome sequencing project, and human genomic PCR primer pairs
for the Washington University Genome Center mapping project, with success rates
exceeding 96% and 81% respectively. It is available for research purposes from
the authors, at no cost, in both text output and interactive graphics (X
C language source code for OSP is available (for research purposes only) at no
cost from the authors, in either the text output version (tested for VAX/VMS,
PC, MAC, and SUN Sparcstations), or interactive X windows graphics version
(tested for SUN Sparcstations).
To obtain OSP please send your postal address either to Phil Green by email,
(pg at genome.wustl.edu) or (preferably) by FAX to (314) 362-2985 c/o Paula,
the secretary handling OSP requests. You must provide a signed licensing
agreement (which she will send you) and a stamped addressed mailer with
diskette before the program can be sent to you. Sorry for the formalities.
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