Lec 6: Gel Electrophoresis

year students
3rd
molecular biology (theoretical)

Dr. Rana Adil Hanoon

Asst. Prof. In Genetic Engineering and Biotechnology
•Electrophoresis is the migration of charged particles or
molecules in a medium under the influence of applied electric
field.

•The electrophoresis machine has an anode (positive end) and a

cathode (negative end), that attract negatively and positively
charged molecules respectively.
▪ Electrophoresis is a molecular technique that separates
nucleic acids and proteins based on:
Size and charge
▪ The term electrophoresis comes from the Greek, and means, "
transport by electricity“.
▪ Electro refers to electron flow or current.
Phoresis refers to movement.
Gel electrophoresis
• A method of separating DNA in a gelatin-like material using an
electrical field
– DNA is negatively charged
– when it’s in an electrical field it moves toward the positive side

The DNA is pulled through the gel because it is negatively

charged, as a result of its phosphate back bone.
• DNA moves in an electrical field…
– so how does that help you compare DNA
fragments?
• Size of DNA fragment affects how far it travels
– small pieces travel farther
– large pieces travel slower

DNA → →→→→→→

– “swimming through Jello”

+
The rate of migration of an ion in electrical field depend on
factors:

1. Net charge of molecule.

2. Size and shape of particle.
3. Strength of electrical field.
4. Properties of supporting medium.
5.Temperature of operation.
Methods of Electrophoresis
All types of electrophoresis are based on the same principle. The difference
between methods is:

1- Support medium (cellulose, polyacrylamide, agarose)

2- Geometries (Vertical and horizontal)
3- Buffers
What is a gel?
A gel is a semi-solid, jelly-like material with small pores that act like a sieve (filter). It
helps separate molecules (like proteins or DNA) based on their: size, and charge.

Gel electrophoresis, is commonly made from a cross-linked polymer such as

Types of Gel:
A. Agarose
B. Polyacrylamide
C. Starch
 Agarose Gel is a polysaccharide, that forms a mesh with tiny holes (porous)
derived from seaweed.

Agarose powder is mixed with water and heated until it dissolves completely
(becomes a clear liquid).

The agarose polymers are flexible and move freely in hot water.
• when the liquid cools, the agarose polymers stick together and form a solid
gel with tiny pores. The size of the pores depends on the agarose
concentration .

• Acts as a sieve for separating molecules.

Concentration affects molecules migration

Low conc. = larger pores.

better resolution of larger DNA fragments.

High conc. = smaller pores.

better resolution of smaller DNA fragments.
Polyacrylamide Gel Electrophoresis (PAGE)

• Acrylamide (monomer) and bis-acrylamide (cross-linker) are mixed,

When polymerized, they form a mesh-like gel with small pores.

• Structure of acrylamide (CH2=CH-CO-NH2).

• Polyacrylamide gel structure held together by covalent cross-links.

• Polyacrylamide gels are tougher than agarose gels.

• Does not interfere with protein movement (no charge), stable, clear easy
to see bands after staining.
Buffers
• Buffers maintain pH and provide ions to support conductivity.

• Most common buffer used is called TRIS (a chemical that controls pH).
– [tris(hydroxymethyl)aminomethane].
• Another compound is added to make Tris an effective buffer
— either boric or acetic acid.
• Another compound is added to bind metals EDTA.
chelates the Mg2+ ion which is required for enzyme DNAse as a cofactor
(a chemical that stops DNA-breaking enzymes by removing magnesium ions).

• The buffer is either TBE or TAE.

✓ TBE is made with Tris/Boric Acid/EDTA
✓ TAE is made with Tris/Acetic Acid/ EDTA
The main difference between TBE and TAE, chemically, has to do with
composition.

• TBE is a good choice when you need high resolution for small DNA
fragments.

• TBE buffer has a greater buffering capacity and will give sharper resolution
than TAE buffer.

• TAE is a good choice when working with larger DNA fragments or for
cloning.

• TAE buffer has a lower buffering capacity than TBE buffer.

Material required for agarose
gel electrophoresis.
Power supply (Applied voltage )
• ↑ voltage, ↑ rate of migration.
• The higher voltage, the more quickly the sample
runs.
• But if voltage is too high, gel melts.
• The best separation will apply voltage at no more
than 5V/cm of gel length.

Gel casting trays, which are available in a variety of

sizes and composed of UV transparent plastic.

A Comb
• A comb is placed in the liquid agarose before it has
been poured.
• Removing the comb from the hardened gel
produces a series of wells used to load the DNA
A DNA marker (also known as a 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".
Loading buffer
Because DNA is colorless, it is not immediately visible in
the gel. Therefore, we will add loading buffer to help see
the DNA.

Loading buffer is mixed with the DNA sample before the

mixture is loaded into the wells.
The loading buffer contains :

1- a dense compound, which may be glycerol or sucrose,

that raises the density of the sample so that the DNA
sample may sink to the bottom of the well.

2- The loading buffer also include colored dyes such as

xylene cyanol and bromophenol blue used to monitor the
progress of the electrophoresis.
Staining of DNA
•To visualize the DNA, the gel is stained with a fluorescent dye that binds
to the DNA, and is placed on an ultraviolet transilluminator which will
show up the stained DNA as bright bands.
• UV absorbance maxima at 300 and 360 nm and emission maxima at
590 nm.
• Examples:
• ethidium bromide is mutagenic so care must be taken while handling
the dye.
• Syber safe.
• Gel red.
• Factors affecting resolution
Resolution = separation of fragments
Good resolution = sharp, well-spaced bands
Poor resolution = faded, smeared bands.

Resolution is affected by
✓agarose concentration
✓salt concentration of buffer or sample
✓amount of loaded DNA
✓voltage
Analysis
•The amplified DNA segment can then be observed with techniques
such as gel electrophoresis.
•Results should be compared with both:
• Positive control.
• Negative control.

PCR controls used to monitor effectiveness

Positive control
Indicates that all reaction ingredients are working.
Known sample.
Negative control
Indicates that there is no contaminating DNA in
any of the reaction reagents.
Includes all reaction ingredients except template
DNA.
Types of Electrophoresis
1- Agarose Gel Electrophoresis
• Agarose gel is a highly purified uncharged polysaccharide derived from agar.
• It is prepared by dissolving agarose in boiling water and allowing it to cool to
40 °C.
2- Native – Polyacrylamide gel electrophoresis (PAGEA)

• Native gels are run in non-denaturing conditions.

• Separation is based upon charge, size, and shape of molecules.

• Useful for separation or purification of mixture of proteins.

3- Denatured -PAGE or SDS-PAGE:
• Separation is based upon the molecular weight of proteins.
• The common method for determining :
MW of proteins.
Checking purity of protein samples.

Preparation of proteins:

mixing proteins with two special chemicals:

• SDS - makes all proteins straight and gives them negative charge

• Mercaptoethanol - breaks any connections between protein parts

• Using heat to help these chemicals work.

SODIUM DODECYL SULFATE (SDS-PAGE)

(SDS-PAGE)
• Native protein is unfolded by heating in
the presence of -mercaptoethanol and
SDS.
• SDS binds to the protein.
• Large polypeptides bind more SDS than
small polypeptides, so proteins end up
with negative charge in relation to their
size.
• When treated with SDS and a reducing
agent, the polypeptides become rods of
negative charges.
• Thus, we can separate the proteins based
on their mass.
4- Isoelectric Focusing
• IEF is a technique used to separate proteins based on their isoelectric point (pI).
• The pI the exact pH where a protein has no charge (not positive, not negative).
• Isoelectric focusing prepare by using a gel with a pH gradient (from acidic to basic).
• Protein migrate into the point where its net charge is zero – isoelectric pH.
• When a protein reaches its pI, it stops moving because it has no net charge at that pH.
• The result is distinct protein bands separated by their pI values.
• Proteins move toward the region of the gel where the pH matches their pI.
 5- Two- Dimensional Gel Electrophoresis (2-D ELECTROPHORESIS )
This method used to separate and study many different proteins at the same time. It works in
two steps to sort proteins first by their electric charge, then by their size.

• In the first dimension, proteins are resolved in

according to their isoelectric points (PI) using
immobilized pH gradient electrophoresis (IPGE),
isoelectric focusing (IEF).

• In the second dimension, proteins are separated

according to their approximate molecular weight
using SDS-PAGE.
First Step - isolation by Charge:

• Take a special gel strip that has different acidity levels

(pH) along its length, then put the protein mixture on
this strip.

• They stop moving when they reach the spot where the
pH matches their natural charge (called pI)

• Now all proteins are lined up by their charge.

Second Step - isolation by Size:

• Take the strip from the first step and lay it flat on a
new gel.

• Add a SDS that makes all proteins equally

negative

• When used the electricity, small proteins move fast

and far, and big proteins move slow and stay near
the top, now proteins are separated by size too.
Final Result:

• We get a gel with many dark spots, each spot is a

different protein
• The left-right position shows its charge
• The up-down position shows its size.
6- Pulsed field gel electrophoresis
• is a technique used for the separation of large deoxyribonucleic acid (DNA) molecules by
using electricity that changes direction (periodically changes direction).

• DNA must be cleaved by restriction enzymes (like tiny scissors) to cut DNA into smaller
pieces, then the DNA is placed into a gel.
• Normal gels use one-
direction current, but PFGE
uses pulsed current (switches
directions slowly).

• This helps big DNA clarified

and move forward in the gel.
What do you think can we use
this technique for?
Forensics
Applications of Gels:

• Estimation of the size of DNA and protein molecules.

• Analysis of PCR products, for example in molecular genetic diagnosis or genetic

fingerprinting.

• Separation of restricted genomic DNA or of RNA.

• Examples :
▪ Forensics.
▪ Molecular biology.
▪ Genetics.
▪ Microbiology.
▪ Biochemistry.
The results can be analyzed quantitatively by visualizing the gel with UV light and a gel
imaging device; analyzing the intensity of the band or the measure of the spot of interest.
 DNA fingerprint
• Why is each person’s DNA pattern different?
– Introns & other Non-Coding regulatory Sequences:
• don’t code for proteins
• made up of repeated patterns
– CAT, GCC, and others
– each person may have different number of repeats
• many sites on our 23 chromosomes with
different repeat patterns

GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT
CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA

GCTTGTAACGGCATCATCATCATCATCATCCGGCCTACGCTT
CGAACATTGCCGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA
 DNA patterns for DNA fingerprints
Allele 1 cut sites repeats cut sites
GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT
CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA

Cut the DNA: 3 Fragments

GCTTGTAACG GCCTCATCATCATCGCCG GCCTACGCTT
CGAACATTGCCG GAGTAGTAGTAGCGGCCG GATGCGAA
1 2 3

– DNA → +
allele 1
 Differences between people
Person 1 cut sites cut sites
GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT
CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA

Person 2: more repeats

GCTTGTAACGGCCTCATCATCATCATCATCATCCGGCCTACGCTT
CGAACATTGCCGGAGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA

1 2 3

DNA fingerprint
– DNA → +
person 1

person 2
 Uses: Evolutionary relationships
• Comparing DNA samples from different organisms to measure evolutionary relationships

turtle snake rat squirrel fruitfly

1 2 3 4 5 3 4
–
DNA
1 2 5

+
 Uses: Medical diagnostic

• Comparing normal allele to disease allele

chromosome chromosome with

with normal disease-causing
allele 1 allele 2 –
DNA


Example: test for Huntington’s disease

+
 Uses: Forensics

• Comparing DNA sample from crime scene with suspects &

victim

suspects crime
scene
S1 S2 S3 V sample
–DNA


+
 Uses: Paternity
• Who’s the father?

Mom F1 F2 child
–
DNA
