NAME
    Bio::MCPrimers

DESCRIPTION
    Creates molecular cloning PCR primer pairs for a given gene so that the
    gene can be directionally inserted into a vector. Solver is generic,
    restriction enzymes and their order in the vector are specified in the
    caller.

EXPORT SUBROUTINES
    sub find_mc_primers

        $orf,            # ATGC string (I use 21 NT upstream, 200 NT downsteam)
        $flag_hr,        # anonymous hash reference to flags from mcprimers.pl
        $pr3_hr,         # hash reference to Primer3 Boulder file tags
        $ecut_loc_ar,    # enzyme cut location array reference from caller
        $vcut_loc_ar,    # vector cut location array reference from caller
        @re              # array of restriction enzyme strings from caller
    
    Not explicitily exported. Use Bio::MCPrimers::find_mc_primers

    See mcprimers.pl for an example of use and front-end.

INSTALLATION
        MCPrimers.pm     - Place into lib/Bio/MCPrimers.pm
        CloningVector.pm - Place into lib/Bio/Data/Plasmid/CloningVector.pm
        Vector files     - Make vector file directory accessable
        mcprimers.pl     - Place in a directory where it can be accessed 
        mcprimers_gui.pl - Place in a directory where it can be accessed 
        mcprimers.acd    - Put in acd directory for EMBOSS 
                           Only checked with acdvalidate 
    
        MCPrimers_manual.doc    - User documentation
    
        MCPRIMER_DIR     - Set this environment variable to point to the 
                           directory containing mcprimers.pl  
        PRIMER3_DIR      - Set environment variable to point to Primer3 
                           executable directory.
    
        MSWindows - use primer3.exe
        Other     - use primer3_core
 
DEPENDENCIES
    Primer3 used as primer3.exe on MSWindows and as primer3_core otherwise.

    Used by mcprimers.pl, which is used by mcprimers_gui.pl:

        Bio::MCPrimers,
        Bio::Data::Plasmid::CloningVector.pm

    Used by mcprimers_gui.pl:

        Tk
        IPC::Open3
        Tk::FileSelect
        Tk::ROText

SYNOPSIS
    mcprimers.pl -help mcprimers.pl -vectorfile pet-32a.txt cyss.fa cyss.pr3
    mcprimers_gui.pl

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

    See mcprimers.pl for an example of the use of Bio::MCPrimers itself

    Note: mcprimers.pl is a command line program mcprimers_gui.pl is a GUI
    on top of mcprimers.pl

LIMITATIONS and TROUBLESHOOTING
    See MCPrimers_manual.doc

    Note: Runs use intermediate files keyed to PID.

BUGS
    Probably. Use at your own risk.

    This software comes with no guarantee of usefulness. Use at your own
    risk. Check any solutions you obtain. Stephen G. Lenk assumes no
    responsibility for the use of this software.

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.

    Primer3 is Copyright (c) 1996,1997,1998,1999,2000,2001,2004 Whitehead
    Institute for Biomedical Research. All rights reserved.

    CloningVector (C) Tim Wiggin and Steve lenk 2006

AUTHOR
    Stephen G. Lenk, November 2005, 2006.

    slenk@emich.edu

ACKNOWLEDGEMENTS
    Primer3 is called by this code to verify that the PCR primers are OK.

    Thanks to Tim Wiggin for good ideas at the start. Tim rewrote
    CloningVector.pm in 2006

    Thanks to Dan Clemans for showing me molecular cloning in the first
    place. I am using Dr. Clemans's ideas about good MC primers in this
    code. Any errors in interpretation or implementation are mine.

    Patricia Sinawe found that earlier versions of MCPrimers did not detect
    out-of-frame solutions and suggested that extra binding sites could be
    included.

    Ken Youens-Clark has provided guidance in the proper naming of this
    software so that it functions cooperatively with other Perl modules.

    Anar Khan and Alastair Kerr for their advice at BOSC 2006 regarding
    EMBOSS compatability, Primer3 parameters, and selective use of sites.

    Other references:

    (1) http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

    (2) http://www.mcb.uct.ac.za/pcroptim.htm

