Alternate Cloning Strategies:
Directional cloning:
Subcloning of insert fragments that have different ends. Vector is cut with two enzymes.
Inserts can only go in one direction.
Challenge is finding ends that work the way you want them to, and doing a double digest
with enzymes that may not work under the same conditions (buffer, temp etc)
Non-directional cloning:
Subcloning of insert fragments that have the same sticky ends. Vector is cut with a single
enzyme. Insert can go in either direction.
Use restriction analysis of plasmids obtained to distinguish which direction the insert has
gone in. Aim to cut once or twice in the backbone (vector) and once, eccentrically, in the
insert. (a cut in the center of the insert will not give you directional information, nor will
just popping the insert out with the same enzymes used to insert it)
Another problem with non-directional cloning is that the vector can easily close up on
itself (self-ligate). Because both cut ends are the same, they come together often intramolecular collisions are likely) and if ligase is present, they re-ligate.
One way to deal with this is to dephosphorylate the ends. Using CIP (Calf intestinal
phosphates) or SAP (Shrimp alkaline phosphatase), we can remove the phosphate group
from the 5’ end of each strand. This makes it impossible for ligase to connect that strand
to another. A CIP’d vector has two cuts close together, and is unlikely to hold together
well enough to be transformed. When there is insert present (which has 5’ phosphates)
the ligated vector has a break in each strand, but the circular DNA is held together with
many H-bonds, and can be transformed – the bacterial repair and replicate across the
breaks, until they are irrelevant.
The CIP or SAP MUST be inactivated before insert is added, because if the insert is
dephosphorylated, it will not be ligatable. SAP is heat sensitive – 20 minutes at 65 C
should inactivate it. CIP is not heat sensitive. It is necessary to use phenol/chloroform
extraction to denature it. One then recovers the DNA by Ethanol precipitation. (No need
to CIP in directional cloning because the cut ends are not going to anneal back together if
they are different enzymes.
Compatible ends: Sometimes you can use one enzyme to cut out a fragment, and put it
into a different site because the sticky ends are compatible. Consider
BamHI G’GATCC
And
Bgl II A’GATCT
These are clearly different sites, but because when they are cut, they have the same sticky
end (the ‘ indicates cut site on the upper strand)
The “Other Project” -- illustrates a couple of things:
(fusion proteins, and one way to deal with compatible ends)
Goal: subclone the EGFP (enhanced Green Fluorescent Protein) coding sequence from
pEGFP-N1 into pUC 19
In the pEGFP-N1, the gene for EGFP is behind a mammalian promoter (CMV, or
cytomegalovirus), but we would like to get it behind a bacterial one.
The pUC19 polylinker is at the N-end (that is, toward the AUG) of the coding region for
b-galactosidase (the lacZ gene). There is a bacterial promoter upstream, which is how
lacZ gets expressed for blue-white screening,
If we can pull out the EGFP coding sequence and plug it into the MCS, we may be able
to arrange it so that the reading frame (three nucleotides to one amino acids) continues
sensibly from the small bit of -gal sequence, through some nucleotides in the MCS, and
ends up in the correct reading frame for EGFP. In that case the bacteria should make a
fusion protein, one that has a little bit of lacZ on the N terminal end, a few amino acids
that are derived form the MCS (also called “cloning vehicle) and the whole sequence of
EGFP, from the AUG start to the stop codon)
Good thing: the N end of EGFP is proximal to the MCS of that vector – that
gives us several options of RE to use to try to achieve the proper reading frame.
Problem: at the other end (the C-terminal, or “stop” end) the only enzyme we can
use is the Not I site. The reason that is bad is that there is no Not 1 site in pUC19.
So what can we do?
Here is the plan:
Cut the pEGFP with Not 1
Incubate with dNTP and Klenow, to fill in the ends, and make them blunt
Klenow is the subunit of DNA polymerase that has the polymerase activity but
lacks some of the other “proofreading” activities” of that complex “holoenzyme”
dNTP is a mixture of the triphosphate form of the DNA bases, that are used as
substrate to make new DNA chains
Inactivate the Klenow (it is quite heat sensitive, so 20 minutes at 65 C should do it)
(very important, lest it fill in when we cut with our second enzyme)
Cut with BamHI. This is on the MCS side of the EGFP. The resulting fragments are 730
bp of EGFP, and the other large fragment (backbone of the parent vector) that is large
and we don’t care about. The 730 bp fragment has one BamH1 end, and one blunt end.
Gel purify (run on a gel and cut out) and clean up this fragment
Now we cut the receiving vector, pUC19 with BamH1 and Sma I. Sma cuts its
recognition sequence squarely in half, creating blunt ends. This should allow us to ligate
our fragment in.