LECTURE 5:
Separation and Visualization of
Restriction Fragments and PCR
Amplified DNA
Biotechnology; 3 Credit hours
Atta-ur-Rahman School of Applied Biosciences (ASAB)
National University of Sciences and Technology (NUST)
Gel Electrophoresis
• Restriction enzyme digestions and other
manipulations of DNA enable the results to be
directly visualized.
• Gel electrophoresis is a technique for separating DNA
fragments by size and visualizing them after staining.
• It separates molecules on the basis of their rate of
movement through a gel under the influence of an
electrical field
• Gelatinous agarose is used as the medium through
which DNA passes
• Ethidium bromide is used for staining
• DNA is negatively charged.
• When placed in an electrical field, DNA will migrate
toward the positive pole (anode).
• An agarose gel is used to slow the movement of DNA
and separate by size.
-
Power
++
How fast will the DNA migrate?
•
•
•
•
Strength of the electrical field, buffer, density of agarose gel
Size of the DNA
Small DNA move faster than large DNA
Gel electrophoresis separates DNA according to size
DNA
small
large
-
Power
+
Within an agarose gel, linear DNA migrate inversely
proportional to the log10 of their molecular weight.
Gel Electrophoresis
Agarose is a linear polymer extracted
from seaweed
D-galactose
3,6-anhydro
L-galactose
• Agarose is a polysaccharide obtained from agar.
• Agar is a gelatinous substance derived from a polysaccharide in red algae, where it
accumulates in the cell walls of agarophyte and serve as the primary structural support
for the algae's cell walls
• Agarose forms an inert matrix utilized in separation techniques.
• Polymerized agarose is porous, allowing for
the movement of DNA
Agarose Gel
Scanning Electron
Micrograph of
Agarose Gel (1×1 µm)
DNA Ladder Standard
 12,000 bp
 5,000
DNA
migration
bromophenol blue
+
 2,000
 1,650
 1,000
 850
 650
 500
 400
 300
 200
 100
Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the
sizes of unknown DNAs. Note: bromophenol blue migrates at approximately the same
rate as a 300 bp DNA molecule
Staining of Agarose Gel
• DNA by itself is not visible in gel
• Ethidium bromide is usually added to make
the DNA band visible
• Ethidium bromide molecule interclate
between the bases causing the DNA to
fluorescence orange when the gel is
illuminated with UV light
• Methylene blue is also used
• Most labs use ethidium bromide as it is very
sensitive
Staining of Agarose Gel
Ethidium Bromide
Ethidium binds by inserting itself bewteen the stacked bases
in double-stranded DNA. The ring structure of ethidium is
hydrophobic and resembles the rings of the bases in DNA.
Ethidium is capable of forming close van der Walls contacts
with the base pairs and that's why it binds to the
hydrophobic interior of the DNA molecule.