PRIMER 0.5: Questions and Answers What exactly is PRIMER? PRIMER is a computer program for automatically selecting PCR primers. PRIMER tests oligos for annealing temperature, complementarity to genomic repeat sequences (e.g. Alu), ability to form primer-dimer, and other criteria. Importantly, PRIMER's annealing temperature calculation is based on thermodynamic parameters (with the base stacking term dominant), and is far more accurate than calculations based on GC/AT ratios. When presented with a short DNA sequence, PRIMER can often design a working PCR assay with known PCR product size without any human guesswork. While PRIMER was specifically designed to select primers from cloned and sequenced genomic DNAs, many of the tests it performs are useful for selecting oligos for other types of experiments as well, including other PCR applications, oligo hybridization and dideoxy sequencing. PRIMER was written by Steve Lincoln, Mark Daly, and Eric Lander at the MIT Center for Genome Research. Version 0.5 of PRIMER is fairly reliable, and is available now. While previous prelimary versions had various version numbers, 0.5 is in fact the most up to date version. What types of computers does PRIMER run on? In short: just about anything which supports a standard C language compiler, including: (1) IBM PC and compatibles. A PC/AT (286) class machine or better with 640K of memory and a math coprocessor are required. PRIMER reads and writes ASCII text files which can be written, read, and printed by most standard PC software. (2) Apple Macintosh computers. A Mac II series or SE/30 with a math coprocessor (IIsi and LC owners note this!) and 2Mb FREE memory are required. The present version of PRIMER is not a Macintosh program per se, but provides only a simple text-only interface which happens to run on the Mac. PRIMER reads and writes Mac TEXT files which can be written, read, and printed by many Mac text editors and word processors. (3) Most Unix workstations including Sun SPARCStation and DEC DECStation systems with their standard C compilers. (4) DEC VAX/VMS computers under VMS Version 5 with VAX C. How do I get PRIMER? PRIMER is available from a number of sources: (A) You can get PRIMER from another user, provided you strictly follow the terms listed below and included with the program. We expect PRIMER to be mostly distributed this way. (B) You can download the most recent version off the Internet via anonymous FTP to "genome.wi.edu" (internet number 192.70.250.135). Fetch and read the file README for details. If this all sounds like gibberish, please ask a computer specialist for help - the process of receiving free programs off the network is actually quite straightforward and commonplace to those familiar with network access and some programming. Note that only the C source code is available by FTP, which you must then compile yourself using your C compiler. (C) You can send a request to: "PRIMER c/o The Lander Lab, Whitehead Institute/MIT 9 Cambridge Center, Cambridge, MA 02142. Email to "primer@genome.wi.edu" (Internet), or FAX to 617-258-6505 Request a copy of PRIMER, specifying a 3 1/2" floppy disk for either SPARCStation, PC, or Mac. All disks contain the entire C source code, which should compile and run on any machine listed above, as well as a ready-to-run version for the appropriate machine (Mac or PC). We cannot send out other formats at this time. Please do NOT send money or a blank floppy. Do I really need to compile PRIMER myself, and if so, how? If you receive a ready-to-run version from us or another user, then you do not. If you receive the C source code, or which to make the source code run on a different type of computer, then yes. The C source code we distribute may be compiled using: (1) Microsoft C 4.0 on IBM PC compatibles. The file MAKEPC.BAT included executes the necessary commands. Later versions of Microsoft C may also work. (2) Apple Macintosh computers with ThinkC 4.0.1. A ThinkC project file has been included on the Mac disk. (3) DEC VAX/VMS computers under VAX C with VMS 5.x. The file MAKEVAX.COM included executes the necessary commands, although you may have to change the linking procedure depending on your system's configuration. (4) Unix workstations including Sun SPARCStation and DEC DECStation Systems with the standard Unix C compilers. A Unix "makefile" has been included. Before compiling the program, you may need to modify configuration information in the file "primer.h", included with PRIMER. Read that file for further details. Also, after compiling PRIMER, you should test it, by running the sample.seq sequence through (using standard.cri and mouse.rep), and comparing the output to the test.* files, included with PRIMER. What's the fine print? PRIMER and its documentation are copyright 1991 by the Whitehead Institute for Biomedical Research. All rights are reserved. PRIMER is available for free use for non-commercial purposes by basic research labs in both academia and industry, providing users register using the form supplied with the program. PRIMER may NOT be used to design oligos for any commercial purpose whatsoever (for example: the design or manufacture of oligonucleotides for profit, or for use in a test or kit supplied or performed for profit) without specific written permission from The Whitehead Institute. PRIMER may be freely redistributed to other scientists, provided that all of the source, data, and documentation files ares distributed unmodified. PRIMER may NOT be posted to any bulletin board, included in any shareware distributions, or the like, without specific written permission from The Whitehead Institute. The PRIMER source code may be customized by individual users, although such versions may not be redistributed without specific written permission from The Whitehead Institute. Primer is designed to select primers for PCR applications, although it may may also be useful for designing oligos for other purposes as well. PCR is covered by US patents issued to Cetus corporation, and PRIMER is intended for use with PCR based laboratory procedures only under appropriate license. PRIMER is supplied without any warranty or guaranteed support whatsoever. The Whitehead Institute can not be responsible for its use, misuse, or performance. WHAT ARE ALL THESE FILES WHICH CAME WITH PRIMER? They are: File Description ---- ----------- primer.c PRIMER C Source Code text.c PRIMER C Source Code primer.h PRIMER C Source Code sample.seq Sample Input Sequence File sample2.seq Longer Sample Input Sequence File standard.cri Sample PCR Primer Criteria File sample.rep Sample Repeat Sequence File (Human Alu only) mouse.rep Sample Repeat Sequence File (Mouse Sequences) rat.rep Sample Repeat Sequence File (Rat Sequences) human.rep Sample Repeat Sequence File (Human Sequences) manual.txt PRIMER Manual text file (no line breaks) manual.asc PRIMER Manual text file (with line breaks) readme.txt This Document (text file, no line breaks) readme.asc This Document (text file, with line breaks) makepc.bat DOS Batch file to compile PRIMER makevax.com VAX/VMS Command file to compile PRIMER makefile Unix Makefile to compile PRIMER Other files which may be included with specific versions include: File Description ---- ----------- primer PRIMER Executable for Sun SPARCStations primer.exe PRIMER Executable for PC Compatibles PRIMER PRIMER Executable for Macintosh PRIMER Manual PRIMER Manual (Mac Microsoft Word 4.0 Format) The PRIMER Manual PRIMER Manual (Macintosh Executable) PRIMER README This Document (Mac Microsoft Word 4.0 Format) The PRIMER README This Document (Macintosh Executable) Remember, to legally redistribute PRIMER to anyone else, you must give them unmodified copies of all of the above files (at least those in the top list). ------------------------------------------- Steve Lincoln and Mark Daly MIT Center for Genome Research Whitehead Institute for Biomedical Research 9 Cambridge Center, Cambridge, MA 02142 primer@genome.wi.edu, FAX: 617-258-6505