Biology Reference
In-Depth Information
contain useful information on restriction endonuclease activity and their appro-
priate reaction conditions.
5.7 Joining DNA Molecules
Different DNA fragments cleaved by restriction endonucleases can be joined
by ligases. Ligases make it possible to insert exogenous DNA into plasmid vec-
tors. Two
DNA ligases
are commonly used. One DNA ligase is derived from
E. coli
and the other DNA ligase is from the bacteriophage T4. Their require-
ments for cofactors differ. T4 ligase requires ATP, whereas
E. coli
ligase requires
NAD
+
. Both catalyze the joining of a 5
′
-phosphate and a 3
′
-OH group to form
a phosphodiester bond. T4 DNA ligase will catalyze the joining of blunt-ended
DNA molecules and cohesive-ended molecules, although more enzyme is
required for blunt-ended ligation of DNA molecules.
If the restriction endonuclease used generated DNA fragment ends with
uneven ends or overhangs (
Table 5.4
), then the sequences of the DNA within
the single-stranded regions of the two molecules have to be complementary for
ligation to occur. Ligation of four-base extensions is easier than ligation of two-
base extensions. Extensions that consist of G
+
C bases ligate more readily than
those with A
+
T base pairs. [Can you explain why?]
Blunt ends are more difficult to ligate, requiring 20-100 times more T4 DNA
ligase and higher DNA concentrations. The surrounding DNA sequences do not
affect ligation efficiency, but ligation is negatively influenced by the presence of
contaminating endonucleases or by phosphatase.
The optimum temperature for ligating DNA is 37°C, but the hydrogen-bonded
joint between sticky ends is unstable at this temperature. As a result, the liga-
tion reaction is carried out at a temperature that is a compromise between the
optimum for the rate of the enzyme action and the association of the termini,
usually between 4 and 15°C. Ligation reactions often are allowed to take place
overnight at these low temperatures. The ligation reaction can be promoted by
adjusting the ratio of insert DNA and vector DNA. When a linear DNA fragment
is produced by a restriction endonuclease from a circular vector, the linear frag-
ment will often recircularize and hydrogen bond to itself, or to other linear vec-
tor sequences. To prevent this, the linearized plasmid vector DNA can be treated
with alkaline phosphatase to remove 5
′
-terminal phosphate groups. Alkaline
phosphatase prevents recircularization of the plasmid or formation of plasmid
dimers, although the phosphatase must be eliminated if the vector and linear
DNA are to be ligated. Circularization of the vector will then occur if the foreign
DNA (untreated with phosphatase) joins the ends of the vector.
