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Bio-MCPrimers-2.5
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/ Bio::MCPrimers

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 See MCPrimers_manual.doc for user documentation

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. They also insisted that I enhance MCPrimers to do eukaryotic organisms.

Other references:

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

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