Primer design

francis at NCBI.NLM.NIH.GOV francis at NCBI.NLM.NIH.GOV
Fri Oct 20 12:35:18 EST 1995

> From: kilpatri at vms.cis.pitt.edu
> Subject: Primer design

>   I apologize if this is a FAQ - I've just joined and didn't see a
> FAQL posted.

Yes, it is a FAQ, and it is on the FAQ, *but* not of this newsgroup ;-)
It's in the methods newsgroup (bionet.molbio.methds-reagnts), and it's 
from Paul Hengen (of TIBS fame).

The complete FAQ is at ftp.ncifcrf.gov (see below) and I include the
part you want  (even further below).



| B.F. Francis Ouellette  
| GenBank
| francis at ncbi.nlm.nih.gov   

1.  Where can I find the FAQ list for bionet.molbio.methds-reagnts?
This file can be retrieved by FTP or read through a WWW hypertext link such
as Mosaic. It is available by anonymous FTP from ftp.ncifcrf.gov as the file
pub/methods/FAQlist, by anonymous FTP from net.bio.net as the file
http://www-lmmb.ncifcrf.gov/~pnh/, or upon e-mail request to
pnh at ncifcrf.gov
My intention for creating this FAQ list is not to attempt a comprehensive
review of the subjects discussed within the newsgroup bionet.molbio.methds-
reagnts, but rather to provide a quick resource for first time users. Many
of the questions answered below are asked by people new to the group on a
recurring basis.
Citations of this file may be made as:
Hengen, Paul N. (1995) "Frequently Asked Question (FAQ) list for
bionet.molbio.methds-reagnts" version number NN.DD.MM.19YY * available via
anonymous FTP from ftp.ncifcrf.gov as file pub/methods/FAQlist or upon request
by e-mail to pnh at ncifcrf.gov
* The version number is designated as: version.day.month.year
  [NN=number, DD=day, MM=month, and YY=year].
If anyone would like to make additions, corrections, or suggestions to
improve this FAQ list, please write to me at pnh at ncifcrf.gov

>   I'm interested in finding a program for PCR primer design that will
> run on IBM compatibles (Windows preferred, DOS may be OK). Inexpensive
> programs are preferable, of course, but any help would be greatly
> appreciated. Thank you!

19. How should I select a set of primers to use for PCR?
See: Innis, M. A., and D. H. Gelfand. 1990. Optimization of PCRs. In:
PCR Protocols: A guide to methods and applications. Academic Press,
New York.
Here are some general pointers:
1. Try to keep the primer 50% G-C give or take 15%. If overly G-C rich add 
a string of As or Ts at 5' end; If overly A-T rich, do the same with Gs and Ts.
2. Try to avoid Gs and Cs at 3' end of the primers. This may increase
the chance of forming primer dimers.
3. Avoid self-annealing regions within each primer.
4. Compute Tm as sum of 4 C for G/C and 2 for A/T, then subtract 5 C 
from this value and that is our annealing temp. Naturally, the annealing 
temp will be that of the primer with the lower value. Differences of 4-6 C 
do not seem to affect yield of PCR. Ideally you would like the Tm for each
primer to match and be within the 70-75 degrees C range.
5. A good practice is to check the target DNA sequence if it is known
for mispriming areas. A quick check scanning the sequence of vector for
approximately 70% and above homolgy regions can help prevent obtaining
multiple contaminating bands in your PCR.
6. Use of a computer program may help eliminate the use of a poorly
designed pair of primers.
20. 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
language compiler.
- 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,
          Whitehead Institute/MIT
          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
distribution/primer.0.4 directory.
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
windows) versions.
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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