The ligation of DNA fragments is explained in this section
Aim of the experiment: To perform the ligation of linearized T-vector with DNA fragment or the ligation of any restriction enzyme digested DNA fragments using T4
PRINCIPLE OF LIGATION OF DNA FRAGMENTS
The basic strategy in molecular cloning is to insert a DNA fragment of
interest (a segment of DNA) into a DNA molecule (called a vector) that is
capable of independent replication in a host cell. The result is a recombinant
molecule composed of the DNA insert linked to vector DNA sequences.
Construction of these recombinant DNA molecules is dependent on the
ability to covalently seal single stranded nicks in DNA. This process is
accomplished both in vivo and in vitro by the enzyme DNA ligase. DNA
ligation is the process of joining together two DNA molecules ends (either
from the same or different molecules). The enzyme that joins the DNA
fragments is called DNA ligases. The DNA ligase seals the nicks in DNA by
formation of phosphodiester bond between adjacent 3’ hydroxyl and 5’
phosphate termini. The enzyme extensively used in joining DNA fragments
is T4 DNA ligase. The ligase joins both cohesive end as well as blunt ended
DNA. It is a single polypeptide with a M.W of 68,000 Dalton requiring ATP
as energy source. The maximal activity pH range is 7.5-8.0. The enzyme
exhibits 40% of its activity at pH 6.9 and 65% at pH 8.3. The DNA fragment
(PCR product) has an extra ‘A’ at 3’ end so that it can complementally bind
to the ‘T’ at the 5’ end of the T- vector.
Illustration of ligation experiment
MATERIALS REQUIRED FOR LIGATION OF DNA FRAGMENTS
- Restriction digested T-vector and PCR product (DNA)
- T4 DNA ligase
- Ligation buffer
- Nuclease free distilled water (autoclaved)
- Gel loading dye
- Ethidium Bromide
- Micro tips
- 50x TAE buffer
- Electrophoresis unit and power supply
- Microwave oven/heater
- UV transilluminator
PROCEDURE FOR LIGATION OF DNA FRAGMENTS
- Three separate vials are taken and are labelled as
reaction, +ve control and –ve control.
- 5µl of PCR product of DNA fragment is added to
reaction and –ve control vials only.
- 1 µl of 10X Ligation buffer is added to each of the three
- 1 μl of T4 DNA Ligase (1 U) is added to reaction and
+ve control vials only.
- 5 μl, 8 μl, 7.5 μl of water is added to reaction, +ve
control and –ve control vials, respectively.
- The total volume in each of the vials is 10 μl. Incubate
for 1 hr at 37ο
- The prepared mixtures can be analyzed in bacterial
transformation in bacterial cells or they can be analyzed
onto agarose gel.
Results and Discussion:
References: DEPARTMENT OF BIOTECHNOLOGY, SCHOOL OF BIOENGINEERING, SRM UNIVERSITY