epitope tag: two or one?
Frank O. Fackelmayer
fof1 at chclu.chemie.uni-konstanz.de
Mon Jun 22 09:50:38 EST 1998
Chris Boyd wrote:
> rmeili_nospamare at hotmail.com wrote:
> : Thanks for your answers:
> : I was hoping for a more than linear effect for two vs one epitope which
> : seems not to be the case
> According to rumour, sometimes the effect _is_ greater than linear. I
> haven't seen this supported in published data, though. And the more
> tags you add, the more likely you are to mess up the protein's
> structure and function.
> : Chris Boyd and Darren Natale: It is cheap to add one epitope but it will be
> : more difficult to add multiple epitopes by PCR alone.
> Why? You can add 4 HA tags to the C-terminus in one go with a primer
> of only (eg) 20 [for initial priming] + 4*27 [the tags] + "TGAATTC"
> [stop codon + EcoRI site] + 1 [for luck] = 132 nt: even long oligos
> are not expensive these days. You can eliminate the possibility of
> mispriming by creative choice of wobble position bases in the tags.
> Admittedly, after PCR you MUST sequence to verify the constructs, but I
> still think it's more cost-effective than using tagging vectors (except
> perhaps for one-offs).
I wouldn´t trust oligos of more than 100nt. The chemistry of oligo synthesis
is at its limits there, no matter how good the chemist is who makes the oligo.
As an alternative, I would recommend you order two complementary oligos
encoding ONE tag, with restriction site overlaps at the ends. To introduce
one tag, anneal the oligos by 5´ 95C, 5´ 70C, and slow cooling to RT, then
clone into the vector linearized with the chosen enzyme (but NOT
dephosphorylated). Use a 1000fold molar excess of annealed primers, and you´ll
end up with over 80% positive clones (i.e. with one copy inserted; inserts
will be in both possible orientations). Directional cloning is, of course,
For more than one tag, phosphorylate both primers before annealing (or order
them phosphorylated), then follow the above protocol. Directional cloning is
almost a must to ensure the oligos are inserted in the wanted orientation
only. You´ll end up with some clones having 1 copy, but also clones with
several copies of the insert, encoding several tags. Select one that has the
number of inserts you want. You´ll need only two relatively short oligos. Very
cheap, extremely effective.
Hope this helps,
More information about the Methods