README for MCPrimers.pm V2.1, mcprimers.pl, and CloningVector.pm

1. Authorship and copyright

Author:    Stephen G. Lenk, November 2005, May 2006.
Copyright: Stephen G. Lenk (C) 2005, 2006. All rights reserved. 

This program is free software; you can redistribute it and/or  
modify it under the same terms as Perl itself.
Licenced under the Artistic Licence.

############################################################################
# This software comes with no guarantee of usefulness.
# Use at your own risk.
# There is no guarantee this code will find the best solution, or even any 
# solution, or that the solutions it finds will be correct or useful to 
# you.
#
# Use at your risk. Check any solutions you obtain.
#
# Stephen G. Lenk assumes no responsibility for the use of this software.
############################################################################

Primer3 is called by this code to verify that the PCR primers are OK.
Primer3 is Copyright (c) 1996,1997,1998,1999,2000,2001,2004
Whitehead Institute for Biomedical Research. All rights reserved.

2. Installation

MCPrimers.pm     - Place into Bio/MCPrimers.pm
CloningVector.pm - Place into Bio/Data/Plasmid/CloningVector.pm
mcprimers.pl     - Front end script. Make available to users.

PRIMER3DIR -   Set this environment variable to point to diectory containing
               Primer3 executable.

If PRIMER3DIR is not set, MCPrimers will set it to '.' by default.

MSWindows -    use primer3.exe
Other OS  -    use primer3_core

The code can be run in the download directory if an installation is not
desired.

3. Purpose

Creates molecular cloning PCR primer pairs for a given gene so that the
gene can be directionally inserted into a vector. pET-32a is the only
vector currently supported. Solver is generic, restriction enzymes and 
their order in the vector are specified in mcprimer.pl and in 
CloningVector.pm

4. Use

Use:     perl mcprimers.pl [options] -vector name dna.fasta > result.pr3
Options: [-h] [-shift search_shift] [-clamp (both | 3prime)]

-h        help
-vector   cloning vector name (i.e. pET-32a)
-shift    integer value to shift search of left RE match into gene
          use -shift 12 (or other value) if initial search fails
-clamp    GC clamp
          'both' will clamp last NT at both ends to G or C (default)
          '3prime' will clamp last NT on 3' ends to G or C
          
Example:  

perl mcprimers.pl -vector pET-32a -shift 12 -clamp 3prime cysS.fa > cysS.pr3

Note:        Use perl -Ilib if the modules are still in the local lib dir.

Limitations: Does not account for redundancy codes in FASTA files.

Note:        These runs use intermediate files. This means that
             CGI or other use that can overwrite these files is
             a bad idea. 

5. Test Results (V2.1)

Passes test.pl clean on Windows XP with Activestate Perl 5.8.7

Passes test.pl clean on OSX with Activestate Perl 5.8.8

Passes test.pl clean on Linux with the standard installed Perl 5.8.7 

Note that HIcysS.fa file needs to be written out in the right text format
prior to running test.pl

OSX with non-Activestate Perl (5.8.1) there are different results each time 
from mcprimers !?

---------------

Enjoy,

Steve Lenk
slenk@emich.edu
May 2006
