Download About DNA Ligase The term ligase comes from the latin ligare

About DNA Ligase
The term ligase comes from the latin ligare, which means “to glue together”. DNA ligase is a
special type of ligase, which is an enzyme that repairs breaks in DNA molecules in the cell.
Ligase is present in many organisms from bacteriophages to humans, and is used to join
Okazaki fragments together. Do you remember what those are? We talked about how DNA can
only replicated from the 5’ end to the 3’ end, so as replication is occurring, it happens
continually on one strand, but not on the other. The bits (about 1000 nucleotides long) that are
replicated on the complementary, anti-parallel strand, called the lagging strand, are Okazaki
fragments.
Applications in molecular biology research
DNA ligase is used in gene cloning to join DNA molecules together with a phosphodiester bond
to fully repair the DNA. (Do you remember identifying the phosphodiester bond in the Jmol
activity?) DNA ligases have become an indispensable tool in modern molecular biology research
for generating recombinant DNA sequences. For example, DNA ligases are used with restriction
enzymes to insert DNA fragments, often genes, into plasmids.
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Here’s an image of DNA ligase
How Does Ligation Work
The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3'
hydroxyl ends of one nucleotide, ("acceptor") with the 5' phosphate end of another ("donor").
(Do you remember identifying the 3’ and 5’ ends of the DNA stick model?)
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DNA sequences of interest can be inserted into a vector, like a plasmid, which are used to make
a recombinant organism. Enzymes called restriction endonucleases cut the DNA into fragments
at particular sites. A common restriction endonuclease is EcoR I which will cut lambda DNA
when it recognizes the sequence AATTC. It cuts it unevenly and the result is an end that has
single nucleotides, which allows complementary sequences, like those that might be targeted
toward creating a recombinant, to bond to the exposed site. These are called “sticky ends”,
DNA can also be cut cleanly across the ladder and those can also be ligated, but it occurs much
more slowly.
DNA Electrophoresis
DNA electrophoresis is an analytical technique used to separate DNA fragments by size. DNA
molecules which are to be analyzed are placed in a gel, and submerged in a buffer that contains
ions, and an electric current is applied to the buffer. Because of the negative, and highly
charged oxygen atoms on the phosphate backbone of DNA, the DNA migrates toward the
cathode. Longer molecules migrate more slowly because they experience more resistance
within the gel. Because the size of the molecule affects its mobility, smaller fragments end up
nearer to the cathode than longer ones in a given period. For larger separations between
similar sized fragments, either the voltage or run time can be increased. Extended runs across a
low voltage gel yield the most accurate resolution.
DNA fragments of different lengths are visualized using a fluorescent dye specific for DNA, such
as ethidium bromide. The gel shows bands corresponding to different DNA molecules
populations with different molecular weight. Fragment size is usually reported in "nucleotides",
"base pairs" or "kb" (for thousands of base pairs) depending upon whether single- or doublestranded DNA has been separated. Fragment size determination is typically done by
comparison to commercially available DNA markers containing linear DNA fragments of known
length.
Objectives:
To ligate fragments of DNA using T4 ligase, and then visualize those fragments using gel
electrophoresis.
Materials
Tube #1: Landda DNA/Hind III Marker 10 μL
Tube #2: Lambda DNA EcoR I, Control, 10 μL
Tube #3: Lamda DNA/EcoR I, Ligated, 10 μL
1 Agarose Gel (0.8%) on Gel Tray
1-5 μL Micropipets
TBE Running Buffer 1x, 350 mL
Power Supply
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Electrophoresis Chamber
Dilute WARD’s QUICKView DNA Stain, 100 mL per gel
Distilled Water
Staining Tray
Spatula
Ligation Buffer 10x
T4 DNA Ligase
Ice Bath
Water Bath
Procedure
1. Add 2 μL ligation buffer 10x and 3 μL T4 DNA ligase to the tube containing 10 μL lambda
DNA/EcoR I digest without the loading dye.
2. Plance the tube in a 16 C ice water baath and incubate for 20 minutes. At this
temperature the T4 DNA ligase catalyzes the ligation of more than 95% of the lambda
DNA fragments.
3. Inactivate the enzyme by incubating the tube in a 65 C hot water bath for 10 minutes.
4. Remove the tube from the hot water bath and add 2 μL of loading dye.
Loading and Running a Gel
1. Load 10 μL from each sample tube onto a corresponding gel lane with a micropipet. Do
not pierce the bottom of the wells with the micropipet, and do not overload the wells.
Lane #1: Lambda DNA/Hind II Marker
Lane #2: Lambda DNA/EcoR I, Control
Lane #3: Lambda DNA/Eco R I, Ligated
Note: The amount of DNA in the reaction tubes is really small. Make sure your
instructor has demonstrated how to transfer samples from the tubes into the wells.
2. Place the loaded gel, on the gel tray, in the center of the chamber. Position the wellside of the gel near the black (negative) electrode.
3. Add approximately 350 mL of 1x TBE running buffer to the chamber: Carefully pur buffer
from a beaker into one compartment of the cell. When the level of the buffer in each
compartment reaches the gel, pour running buffer into the other compartments until
the level of buffer in each compartment reaches the gel. Add more running buffer to
the compartment nearest the red (positive) terminal until the buffer level is
approximately 2 mm above the gel.
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Do not overfill the compartments.
4. Making sure the cover is dry, slide it onto the electrophoresis chamber. Wipe off any
spills on the apparatus before proceeding to the next step.]
5. Making sure that the patch cords attached to the cover, as well as the female plugs and
the banana jacks on the chamber, are completely dry, connect the red patch cord to the
red electrode terminal on the power supply. Connect the black patch cord to the black
electrode terminal on the power supply.
Note: Be sure your instructor checks the connections before proceeding to the next
step.
6. Plug in the power supply and set it to either 75V or 125V.
7. Turn on the power supply. The red power light will illuminate, and bubbles will form
along the platinum electrodes.
8. Observe the migration of the sample down the gel toward the red electrode. Turn off
the power when the loading dye has neared the end of the gel. Unplug the power
supply.
9. Wait approximately 10 seconds, then disconnect the patch cords first from the power
supply and then from the chamber.
10. Put a notch in one side of the gel to ensure the lanes can be idetified after the gel is
removed from the unit.
11. Lifet the gel tray with the gel from the chamber and gently place in the staining tray.
12. Wearing protective gloves, pur approximately 100 mL of warmed dilute stain into the
staining tray so the stain just covers the gel.
13. Cover and let gel stain for approximately 30 minutes.
14. When the gel has completed staining, carefully decant the used stain. Make sure the gel
remains flat and does not move up against the corner. Decant the stain into a sink and
flush with water.
Note: The dilute DNA stain may be saved and reused several times. For best
results, reheat the stain before reusing.
15. Add warm distilled or tap water (50 to 55 C) to the staining tray. To accelerate
destaining, gently rock the tray. Destain until bands are distinct, with little background
color. This will take between 20 and 30 minutes, depending on the amount of agitation.
Change the water several times, or destain the gel in water overnight. Destaining
overnight will produce dark blue DNA bands and a colorless background.
16. View the gel against a light background, such as white paper, or on a light table. Gels
can be stroed in self-sealing plastic bags. For long-term storage, add several drops of
dilute stain the the bag to prevent the DNA bands from fading. If fading does not occur,
the gel can be restained using the above procedure.
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Wash your hands well before leaving the lab.
Analysis
1. Measure the distance of the DNA bands, in millimeters, from the bottom of the sample
well to the bottome of each DNA fragment on the marker. Measuring to the bottom of
each fragment on the marker. Measuring to the bottom of each fragment band ensures
consistency and accurate measurements. Do not measure the migration distance of the
largest fragment mearest the well; it will not be on the standard curve and will skew
results.
2. Record the measurements in Table 1.
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Table 1
DNA Marker Standard
(Lambda DNA/ Hind III)
Standard Curve Plot for Lambda DNA/ Hind III
Fragment Lambda
DNA/Hind III
Length (b.p.)
1
23, 130
2
9, 416
3
6, 557
4
4, 361
5
2, 322
6
2, 027
7
564
8
125
Migration Distance
(mm)
Lambda DNA/Hind
III
Migration Distance
(mm)
Lambda DNA/EcoR I
Lambda
DNA/EcoR I
Length (b.p.)
1. In Graphical Analysis, or on log paper, plot a standard curve for the lambda DNA/Hind III
marker. Plot the migration pattern on the x-axis
2. In Graphical Analysis, or on log paper, plot a standard curve for the lambda DNA/Hind III
marker. Plot the migration pattern on the x-axis
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Standard Curve Plot for Lambda DNA/Hind III
Fragment Length (bp)
100000
10000
1000
10
15
20
25
30
35
40
45
50
55
60
Distance Migrated (mm)
Questions
1. Based on the analysis of the gel, was your ligation reaction successful? Why or why not?
2. Explain the relationship between the bands in lane 2 and lane 3.
3. What might be the reason for the evolution of ligases in bacteria? Specifically, what
adaptive advantage do they give the organism that has them?
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Teacher Notes and Additional Background Information (adapted from Wikipedia)
ATP is required for the ligase reaction, which proceeds in three steps: (1) adenylation (addition
of AMP) of a residue in the active center of the enzyme, pyrophosphate is released; (2) transfer
of the AMP to the 5' phosphate of the so-called donor, formation of a pyrophosphate bond; (3)
formation of a phosphodiester bond between the 5' phosphate of the donor and the 3'
hydroxyl of the acceptor.
In mammals, there are four specific types of ligase.




DNA Ligase: ligates the nascent DNA of the lagging strand after the DNA polymerase I
has removed the RNA primer from the Okazaki fragments.
DNA ligase II: alternatively spliced form of DNA ligase III found in non-dividing cells.
DNA ligase III: complexes with DNA repair protein XRCC1 to aid in sealing DNA during
the process of nucleotide excision repair and recombinant fragments.
DNA ligase IV: complexes with XRCC4. It catalyzes the final step in the non-homologous
end joining DNA double-strand break repair pathway. It is also required for V(D)J
recombination, the process that generates diversity in immunoglobulin and T-cell
receptor loci during immune system development.
Some forms of DNA ligase present in bacteria (usually larger) may require NAD to act as a cofactor, whereas other forms of DNA ligases (usually present in E.coli, and usually smaller) may
require ATP to react. Also, a number of other structures present in the DNA ligase are AMP and
lysine, both of which are important in the ligation process since they create an intermediate
enzyme.
The types of gel most commonly used for DNA electrophoresis are agarose (for relatively long
DNA molecules) and polyacrylamide (for high resolution of short DNA molecules, for example in
DNA sequencing). Capillary electrophoresis has become important for applications such as highthroughput DNA sequencing. Electrophoresis techniques used in the assessment of DNA
damage include alkaline gel electrophoresis and pulsed gel electrophoresis. The measurement
and analysis are mostly done with a specialized gel analysis software. Capillary electrophoresis
results are typically displayed in a trace view called an electropherogram.
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