Analytical Microbiology

MIC402

K.M. Mazharul Alam

Lecturer,
MNS Department,
BRAC University
1
Electrophoretic Techniques

K.M. Mazharul Alam

Lecturer,
MNS Department,
BRAC University
2
Study Outcome

▪ Gel electrophoresis: basic principle and types

▪ Agarose gel electrophoresis
▪ Orthogonal field alternation gel electrophoresis (OFAGE)
▪ Pulsed-field Gel Electrophoresis (PFGE)
▪ Field inversion gel electrophoresis (FIGE)
▪ CHEF (contour clamped homogeneous electric fields)
References

◼ Principles and Techniques of Biochemistry and Molecular Biology (Sixth

edition)
KEITH WILSON & JOHN WALKER

4
Gel Electrophoresis

◼ The term electrophoresis describes the migration of a charged particle under

the influence of an electric field.
◼ Many important biological molecules, such as amino acids, peptides, proteins,
nucleotides and nucleic acids, possess ionizable groups and, therefore, at any
given pH, they exist in solution as electrically charged species either as cations
or anions.
◼ Under the influence of an electric field these charged particles will migrate
either to the cathode or to the anode, depending on the nature of their net
charge.
Gel Electrophoresis
◼ Electrophoresis is usually carried out in aqueous solution.
◼ Electrophoresis involves the use of a gelatinous material such as agarose,
acrylamide, starch or cellulose acetate as the matrix.
◼ The gel acts as a support medium for the sample.
◼ Separation is brought about through molecular sieving technique, based on the
molecular size of the substances. Gel material acts as a "molecular sieve”.
◼ It is important that the support media is electrically neutral.
◼ Commonly used to separate samples containing protein or DNA.
Gel Electrophoresis
◼ 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.

◼ Agar gel is used for separation of different types of protein mixtures as well as
nucleic acids.

◼ Polyacrylamide is most suitable for separation of nucleic acids. It is also

frequently used in separating proteins, peptides and amino acids from microgram
quantities of mixed samples
Gel Electrophoresis

▪ Gel electrophoresis involves an electrical field.

▪ The molecules to be separated are pushed by an electrical field through a
gel that contains small pores
▪ The molecules travel through the pores in the gel at a speed that is
inversely related to their lengths. This means that a small DNA molecule
will travel a greater distance through the gel than will a larger DNA
molecule
▪ DNA and RNA are negatively charged molecules, they will be pulled
toward the positively charged end of the gel.
Why Gel Electrophoresis?

◼ To separate DNA fragments from each other

◼ To determine the sizes of DNA fragments
◼ To determine the presence or amount of DNA
◼ To analyze restriction digestion products
Gel Electrophoresis Classification

◼ Gel Electrophoresis is
carried out in two
methods:
1.Vertical gel
electrophoresis
2.Horizontal gel
electrophoresis
Types Of Denaturation

1. Native: separation by size and charge (charge/mass)

2. Denaturing: separation by size
3. Others: IEF and 2D
Buffer Systems

◼ Continuous system: gel and tank buffers are the same, single phase gel
◼ Examples: agarose and starch gel
◼ Discontinuous system: gel and tank buffers are different, two phase gel
(stacking gel)
▪ Examples: PAGE
Other Types

◼ IEF (Isoelectric Focusing): protein separation based on

isoelectric points in a pH gradient
◼ 2D electrophoresis: combination of IEF and SDS-PAGE
Applications

• Estimation of the size of DNA molecules following restriction enzyme digestion,

e.g. in restriction mapping of cloned DNA.
• Analysis of PCR products, e.g. in molecular genetic diagnosis or
genetic fingerprinting
• Separation of restricted genomic DNA prior to Southern transfer, or of RNA
prior to Northern transfer.
Applications

• Gel electrophoresis is used to provide genetic information in a wide range

of data fields, such as forensics, molecular biology, genetics, microbiology
and biochemistry.
• Human DNA can be analyzed to provide evidence in criminal cases, to
diagnose genetic diseases, and to solve paternity cases.
• Samples can be obtained from any DNA-containing tissue or body fluid,
including cheek cells, blood, skin, hair, and semen.
Agarose Gel Electrophoresis (AGE)
◼ most commonly used in the separation of DNA molecules and so is frequently used
during DNA manipulation techniques, or studies involving identifying individuals
based on their unique DNA sequence.

◼ Agarose gel electrophoresis is a method to separate DNA or RNA molecules by size.

◼ This is achieved by moving negatively charged nucleic acid molecules through an

agarose matrix with an electric field (electrophoresis).

◼ Shorter molecules move faster and migrate faster than longer ones.
AGE: Principle

◼ Agarose gels have lower resolving power for DNA than acrylamide gels. It is
usually used for the separation of DNA fragments of 50 bp-20 kb in size
◼ The pore size of the gel affects the size of the DNA that can be sieved. The
lower the concentration of the gel, the larger the pore size, and the larger the
DNA that can be sieved.
◼ However low-concentration gel (0.1 - 0.2%) are fragile and therefore hard to
handle, and the electrophoresis of large DNA molecules can take several days.
Agarose

◼ Agarose is a polysaccharide obtained from the red

algae 6 Porphyra umbilicalis
◼ Its systematic name is (1 4)-3,6 - anhydro-α-L-
galactopyranosyl-(1,3)-β-D-galactopyranan.
◼ Agarose is a linear polysaccharide (average relative
molecular mass about 12 000) made up of the basic
repeat unit agarobiose, which comprises alternating
units of galactose and 3,6-anhydrogalactose .
◼ Forms a porous matrix as it gels – shifts from random
coil in solution to structure in which chains are
bundled into double helices
Agarose

Red algae Porphyra umbilicalis Electron micrograph of agarose

Why Agarose NOT Agar For Gel Electrophoresis
Of DNA?
Material Required For Agarose Gel
Electrophoresis
◼ Electrophoresis chamber
◼ Agarose gel
◼ Gel casting tray
◼ Buffer
◼ Staining agent (dye)
◼ A comb
◼ DNA ladder
◼ Sample to be separate
Material Required For Agarose Gel Electrophoresis
Buffers
1. During electrophoresis water undergoes hydrolysis : H2O → H+ + OH-
2. Buffers prevent the pH from changing by reacting with the H+ or OH- products
3. Most common buffer used is called TRIS – [tris(hydroxymethyl)aminomethane]
4. Another compound is added to make Tris an effective buffer — either boric or acetic
acid
5. Another compound is added to bind metals: EDTA (Ethylenediamine Tetra-acetic Acid)
6. The buffer is either TBE or TAE
◼ TBE is made with Tris/Boric Acid/EDTA
◼ TAE is made with Tris/Acetic Acid/ EDTA
DNA Ladder

◼ It is a solution of DNA molecules of different

length
◼ DNA Ladder consists of known DNA sizes used
to determine the size of an unknown DNA
sample.
◼ The DNA ladder usually contains regularly
spaced sized samples which when run on an
agarose gel looks like a "ladder".
Staining Of DNA

◼ To make DNA fragments visible after

electrophoresis, the DNA must be stained.
◼ The most common dye used to make DNA or RNA
bands visible for agarose gel electrophoresis is
ethidium bromide (EtBr).
◼ When bound to DNA it fluoresces under ultraviolet
light (reddish –orange colour)
◼ Convenient because it can be added directly to the
gel
◼ Sensitive—detects 0.1ug of DNA
Ethidium Bromide (Etbr)

◼ Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic

acids and allows very convenient detection of DNA fragments in gels.
◼ Inserts itself between the base pairs in the double helix.
◼ When it is exposed to ultraviolet light, ethidium bromide fluoresces. Thus, this
chemical provides both a means of tagging DNA molecules and a means of visualizing
them.
◼ Ethidium bromide is mutagenic so care must be taken while handling the dye.
◼ safer alternatives are available, such as GelRed, which binds to the minor groove.
◼ SYBR Green I is another dsDNA stain. It is more expensive, but 25 times more
sensitive.
Ethidium Bromide (Etbr)
Loading Dye
◼ Bromophenol Blue (Tracking Dye): for visual tracking of DNA migration
during electrophoresis
◼ Glycerol: ensures that the DNA in the ladder and sample forms a layer at
the bottom of the well.
◼ EDTA : included in the solution binds divalent metal ions and inhibits metal-
dependent nucleases.

◼ Buffer: Loading dye is diluted in buffer

Method in Detail
1. Add running buffer, load samples and marker
2. Add running buffer, load samples and marker
3. Run gel at constant voltage until band separation occurs
4. Pour into casting tray with comb and allow to solidify
5. Pour into casting tray with comb and allow to solidify
6. View DNA on UV light box and show results
7. Prepare agarose gel
8. Melt, cool and add Ethidium Bromide. Mix thoroughly.
9. DNA is negatively charged. +- Power DNA • When placed in an electrical field, DNA will migrate toward
the positive pole (anode). H  O2  • An agarose gel is used to slow the movement of DNA and separate by
size.
Analysis

◼ After electrophoresis the gel is illuminated with

an ultraviolet lamp to view the DNA bands.
◼ The ethidium bromide fluoresces reddish-orange
in the presence of DNA.
◼ Photograph it with a digital camera/ with the in-
built camera of the illuminator
Analysis
◼ Although the stained nucleic acid fluoresces
reddish-orange, images are usually shown in
black and white.
◼ Digital photo of the gel.
◼ Lane 1. DNA Markers
◼ Lane 2. empty
◼ Lane 3. a PCR product of just over 500 bases
◼ Lane 4. a PCR product of around 2500 bases
https://youtu.be/hdmQaAycafc
Thank you