The partial chew back
Here's another way to control the ends - by the 3' to 5' exonuclease activity in
the Klenow fragment or T4 DNA polymerase, we can make 5' overhanging ends of controlled
I'll give you an example. Here's the sequence in the region of the multiple cloning
site for the vector pUC18.
If we cut the vector with the enzyme Sma I (CCC^GGG),
which cuts the sequence in only one place, then the two arms will look like this:
Now let's take the products of digestion and add
Klenow fragment and only the substrate dATP. What do we get? It will look like this
when the reaction reaches steady state.
I've marked the 3' terminal A nucleotides in red,
because these will be removed and added over and over again, as long as there is
a large excess of dATP in the reaction.
Now let us set that aside and design polymerase chain reaction primers to isolate
For the oligonucleotide on the left, we design the following (and note the extension
For the oligonucleotide on the right, we design
the following (and note the extension in blue):
Our PCR product using these two oligonucleotides
looks like this, initially:
But now suppose that we add Klenow fragment and
only dTTP as a substrate. We would get this:
Please note that this fits perfectly into our prepared
vector, with a forced orientation:
It is important to note that we didn't have to
touch our PCR product with a restriction enzyme to get these compatible ends. This
is a great relief, because there are often times when our PCR product has an internal
restriction site. If we digest the PCR product with a restriction enzyme, we run
the risk of cutting it into smaller pieces than we had intended.
With this partial chew back method, we might need to treat the PCR product with T4
polynucleotide kinase to apply phosphates to the 5' ends (for efficient cloning with
T4 DNA ligase) but the method is otherwise very straightforward. You have a considerable
amount of control over the ends of DNAs, and can use the design process of PCR to
make compatible ends.
Note: Precise chewing of the ends of the vector requires a large excess of substrate,
perhaps 0.5 mM to 1 mM, and a short reaction time, because you don't want the chewing
reaction to slip beyond the boundary you have set. For example, in the case of preparation
of the vector, what you planned to make is this:
If the dATP concentration is too low, however,
the exonuclease may occasionally remove an A nucleotide and not replace it. You may
start to get this as a product (or worse!):
A better design would have a bit of redundancy
so that a single "slip" of the exonuclease does not ruin the experiment.
Long chew-back methods can be taken to extremes, as in the case of "ligation
independent cloning" described below.