[Bioperl-l] Automatic design of gene fusion primers for pET-32
Stephen Gordon Lenk
slenk at emich.edu
Tue Nov 15 18:59:40 EST 2005
I've written a package to generate correct gene fusion primers for the
pET-32a vector for a given gene sequence. It is flexible enough to use
with other vectors (I believe), but I am working with the pET-32a
vector (Novagen) now. I seek guidance on whether anyone is even
interested in using this software, as the process of designing good
gene fusion primers can be painful.
In case this is unclear, the pET-32a vector allows for the directional
cloning of a gene into a plasmid that can be inserted into a host,
where the plasmid can be turned on to express the inserted gene. A
matched pair of restriction enzymes is used to cut vector and gene,
which are then ligated together. The PCR primers can be used to both
amplify the gene and modify it so that the restriction enzymes will
cut it correctly for proper insertion, where the ideal solution joins
the gene to the plasmid in such a way that the ATG codon joins each at
the front and the downstream join is past the end codon of the gene.
THE WHOLE GENE AND NOTHING BUT THE GENE.
Designing an optimal pair of primers takes time and insight. I have no
desire to prevent such effort, but I have observed that it can be
difficult and frustrating for non-experts. To assist such people, and
for my own amusement, I have writen Perl software to automate this
process. The software uses combinatorics, constraint satisfaction, and
a small bit of expert system flavoring to produce as optimal a
solution as it can. This solution is checked internally against
Primer3 to see if the primers it has desugned are usable. The Primer3
check is automatic and those results are returned to the user, without
striving to discourage later checks. The restriction enzyme pair used
is also returned since this is useful information for those wishing to
do a restriction digest.
Is this of use to anyone? If it is of use, I'd like to appropriately
package and distribute it under the same terms as Perl, that is
without cost to a user, and without liability to myself. Please advise
me as to potential inetrest. I do not plan to Bioperlify it, but it
should be easy to call from a Bioperl environment.
slenk at emich.edu
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