Practical Lessons of Molecular Genetics

Practical lessons of Molecular Genetics
Agarose gel electrophoresis

• Agarose gel electrophoresis is one of the most common
electrophoresis techniques which is relatively simple and
straightforward to perform but possesses great resolving power. The
agarose gel consists of microscopic pores that act as a molecular
sieve that separates molecules based upon the charge, size and
shape.
• Agarose gel electrophoresis is a convenient analytical method for
separating DNA fragments of varying sizes ranging from 100 bp to
25 kb.
• DNA fragments smaller than 100 bp are more effectively separated
using polyacrylamide gel electrophoresis whereas pulse-field gel
electrophoresis is used to separate DNA fragments larger than 25 kb.
Agarose gel electrophoresis can also be used to separate other
charged biomolecules such as RNA and proteins.
Agarose and Polyacrylamide
• Although agarose and polyacrylamide differ greatly
in their physical and chemical structures, they both
make porous gels.
• A porous gel acts as a sieve by retarding or, in some
cases, by completely obstructing the movement of
macromolecules while allowing smaller molecules to
migrate freely.
• By preparing a gel with a restrictive pore size, the
operator can take advantage of molecular size
differences among proteins
Agarose and Polyacrylamide
• Because the pores of an agarose gel are large, agarose
is used to separate macromolecules such as nucleic
acids, large proteins and protein complexes
• Poly acrylamide , which makes a small pore gel, is
used to separate most proteins and small olig
onucleotides.
• Both are relatively electrically neutral
Agarose Gels
• Agarose is a highly purified uncharged polysaccharide
derived from agar
• Agarose dissolves when added to boiling liquid. It
remains in a liquid state until the temperature is lowered
to about 40° C at which point it gels
• The pore size may be predetermined by adjusting the
concentration of agarose in the gel
• Agarose gels are fragile, however. They are actually
hydrocolloids, and they are held together by the formation
of weak hydrogen and hydrophobic bonds
Structure of the Repeating Unit of Agarose, 3,6-
anhydro-L-galactose
Basic
disaccharide
repeating units of
agarose,
G: 1,3-β-d-
galactose
and
A: 1,4-α-l-3,6-
anhydrogalactos
e
Polyacrylamide Gels
• Polyacrylamide gels are tougher than agarose gels
• Acrylamide monomers polymerize into long
chains that are covalently linked by a crosslinker
• Polyacrylamide is chemically complex, as is the
production and use of the gel
Crosslinking Acrylamide Chains
The rate of migration of a DNA molecule
through a gel is determined by the following
parameters :.
❑ Agarose concentration
❑ Size of DNA molecule
❑ DNA conformation
❑ Applied voltage
Agarose concentration:
• The mobility of DNA molecule is inversely
proportional to gel concentration. Higher
percentage gels are sturdier and easier to
handle but the mobility of molecules and
staining will take longer because of the tighter
matrix of the gel. The most common agarose
gel concentration for separating dyes or DNA
fragments is 0.8%. However, some
experiments require agarose gels with a higher
percentage, such as 1% or 1.5%.
Size of DNA molecule
• The sieving properties of the agarose gel
influence the rate at which a molecule
migrates. The separation occurs because
smaller molecules pass through the pores of
the gel more easily than larger ones. If the size
of the two fragments is similar or identical,
they will migrate together in the gel.
DNA conformation
• Different forms of DNA move through the gel
at different rates; DNA molecules having a
more compact shape (e.g. plasmid DNA)
moves faster through gel compared with linear
DNA fragment of the same size. The migration
rate of linear fragments of DNA is inversely
proportional to the log 10 of their size in base
pairs. This means that the smaller the linear
fragment, the faster it migrates through the gel.
Applied voltage
• Mobility of DNA molecule is also affected by
the applied voltage. Within a range, the higher
the applied voltage, the faster the samples
migrate
The protocol can be divided into four
stages
❑Preparation of Agarose gel matrix
❑Sample and marker loading
❑Running the gel at optimum voltage and
optimal time for effective separation
❑Staining the gel for viewing DNA
Preparation of Agarose gel matrix
• The gel is made by dissolving agarose powder in boiling
buffer solution.
• The concentration of agarose in a gel depends on the sizes of
the DNA fragments to be separated, with most gels ranging
between 0.5%-2%. The solution is then cooled to
approximately 55°C and poured into a casting tray which
serves as a mold.
• After the gel solidifies, the gel is submerged in a buffer-filled
electrophoresis chamber The volume of the buffer should not
be greater than 1/3 of the electrophoresis chamber. The most
common gel running buffers are TAE (40 mM Tris-acetate, 1
mM EDTA) and TBE (45 mM Tris-borate, 1 mM EDTA).
solidified agarose gel after removal of
the comb
Sample preparation and loading
▪ Samples are prepared for electrophoresis by mixing
them with loading dyes. Gel loading dye is typically
made at 6X concentration (0.25% bromphenol blue,
0.25% xylene cyanol, 30% glycerol). Loading dyes
used in gel electrophoresis serve three major purposes:
1) add density to the sample, allowing it to sink into the
gel.
2) provide color and simplify the loading process.
3) the dyes move at standard rates through the gel,
allowing for the estimation of the distance that DNA
fragments have migrated.
Loading the DNA sample and marker
into a well in the gel
Running the gel at optimum voltage and
optimal time for effective separation
• A direct current (D.C.) power source is connected to the
electrophoresis apparatus and electrical current is
applied. Charged molecules in the sample enter the gel
through the walls of the wells. Molecules having a net
negative charge migrate towards the positive electrode
(anode) while net positively charged molecules migrate
towards the negative electrode (cathode). The buffer
serves as a conductor of electricity and to control the
pH, which is important to the charge and stability of
biological molecules. Since DNA has a strong negative
charge at neutral pH, it migrates through the gel
towards the positive electrode during electrophoresis
Staining the gel for viewing DNA
• The agarose gel will have to be post stained after
electrophoresis. The most commonly used stain for
visualizing DNA is ethidium bromide (EtBr)*.
• EtBr works by intercalating itself in the DNA molecule
in a concentration-dependent manner. When exposed to
short wave ultraviolet light source (transilluminator),
electrons in the aromatic ring of the ethidium molecule
are activated, which leads to the release of energy
(light) as the electrons return to ground state. This
allows for an estimation of the amount of DNA in any
particular DNA band based on its intensity.
• Etbr is either added to the gel prior to electrophoresis.
Or the gel is stained pos trunning .
▪ Ethidium bromide is a suspect mutagen and carcinogen
so must be handled cautiously. It is a hazardous waste
so must be disposed of according to strict local and/or
state guidelines. Stains containing methylene blue are
considered safer than ethidium bromide, but should still
be handled and disposed with care.
▪ The exact sizes of separated DNA fragments can be

determined by plotting the log of the molecular weight
for the different bands of a DNA standard against the
distance travelled by each band. The DNA standard
contains a mixture of DNA fragments of pre-
determined sizes that can be compared against the
unknown DNA samples.
An image of a gel post electrophoresis

Practical Lessons of Molecular Genetics

Uploaded by

Copyright:

Available Formats

Practical Lessons of Molecular Genetics

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Practical Lessons of Molecular Genetics

Uploaded by

Copyright:

Available Formats

Practical lessons of Molecular Genetics

Agarose gel electrophoresis

▪ The exact sizes of separated DNA fragments can be

You might also like