oligo cloning

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Tue Feb 15 05:12:30 EST 2000


Hi Sarah,

>
> As for the oligo cloning I just thought I would try your method with a PCR product to
> see if it would work.  I thought as you are annealing the two oligo's  together it
> could be the same as having a double stranded PCR product?

well, in principle you are right. The cloning, when seen in molecular terms, works in
the same way (closing a phosphodiester bond between a 5´ phosphate of one DNA molecule
and the 3´ hydroxyl of another). There is one fundamental difference, however, when
thinking of the procedure in practical terms. Oligo cloning works very well because you
can have a huge molar excess of oligos over vector (because the oligo is so small in
comparison to the vector). For cloning a PCR product, it is hardly practicable to use a
2000x excess of fragment over vector (that would be, eg. 10ng of vector and 20ug of
fragment (for simplification vector and insert size presumed to be identical)). Anyway,
it is not necessary to follow that protocol for the cloning of PCR products, as outlined
below.

> Do you do PCR cloning as well ?

Yes, and there is not much magic involved (even though some companies try to make you
believe that). In fact, there are several ways of cloning PCR products. If you can
afford it, and have only a decent number of clonings to do, there are kits available
from several companies to make your life easier. TOPO cloning of PCR fragments is, e.g.,
very convenient and works well most of the times (although there are serious drawbacks
to this method, like the loss of flanking sequences in some cases). If you use Taq
polymerase for amplification (and you really shouldn´t if you are interested in a
faithfully and errorfree amplified sequence!) you can use TA-cloning. This works by
making use of the terminal transferase activity of Taq that often adds a single A to the
3´ end of amplified fragments. This one-base overhang can then be ligated to a one-base
T overhang on the vector. This method also works well, although cloning of one-base
overhangs is usually less efficient the even blunt end cloning and you need VERY good
competent cells. There are also kits available for TA-cloning, but you can quite easily
prepare your own T-vector. A third way for which there is a kit is to use blunt-end
cloning into a vector that has a unique rare-cutter cleavage site. Ligation is done in
presence of the restriction enzyme and results in an enrichment of "correct" clones over
time, because the restriction site is reconstituted in religated (insert-free) vector
but not in vector+insert combinations. Use a proofreading polymerase that makes blunt
ends (Pfu polymerase is great) for this method.

Other more conventional methods of PCR fragment cloning are simple blunt-end cloning
(works only with a proofreading polymerase without terminal transferase activity), or
incorporation of restriction sites into the primers, cutting after amplification, and
cloning into an appropriately cut vector. The second method is preferred for the
construction of protein expression constructs because you have to clone them in-frame
with a purification tag and the fragment does not normally have a restricition site
where you need one... We routinely do that in my lab, so if you need some practical
advice, do not hesitate to contact me via e-mail.

Frank





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