CYTOGENETICS LEC

BSMT 2 - 2ND SEMESTER

ELECTROPHORESIS

Lesson 7: Electrophoresis
testing
Mr. Renz Caburnay 5. Forensics - DNA fingerprinting
ELECTROPHORESIS
PRINCIPLE
● Any experimental technique that is based on movement of
charged particles in electric field in liquid medium.
● Any electrically charged particle dissolved in aqueous solution,
when placed to a constant electric field, will start to migrate
towards the electrode bearing the opposite charge.
○ Ionized solutes move toward either the cathode
(-ve) or the anode (+ve) in an electrophoresis
system.
○ An ampholyte becomes positively charged in a
solution that is more acidic than its isoelectric point
Results after an electrophoretic process (Pi) and migrates towards the cathode.
■ Ampholyte - A particle that is amphoteric.
● Electrophoresis is the separation of charged compounds based
■ Amphoteric - It can act as an acid or a
on their electrical charge.
base.
● It is used analytically to study the properties of a single charged
■ Isoelectric Point - A pH point in which a
species and as a separation technique.
molecule carries any net charge (0).
○ It can be used analytically to study the properties of a single
○ In a more alkaline solution it becomes negatively
charged species or mixtures of molecules.
charged and migrates toward the anode.
○ It can also be used preparatively as a separating technique.
● Electrophoretic mobility - measure of the migration velocity.
● As an analytical tool, electrophoresis is simple, rapid and highly
Significant and characteristic parameter of a charged molecule
sensitive.
or particle.
○ Compared to other processes separating molecules and
● Dependent on the pK values of the charged groups and the
techniques, electrophoresis is the go to for both of the
size of the molecule or particle.
laboratories in the world.
● Influenced by the type, concentration and pH of the buffer,
temperature, and nature of supporting material.
HISTORY OF ELECTROPHORESIS
● The speed of the particle movement will be directly
1937 ● Arne Tiselius first applied electrophoresis to proportional to the applied voltage and particle charge, but
separate samples in a mixture inversely proportional to the particle size.
● Separation of albumin and globulin fractions in
blood serum THREE ELECTROPHORETIC SEPARATION METHODS
● Limited by large sample volumes and a low
electrical field strength ZONE ● Commonly called as
ELECTROPHORESIS Electrophoresis
1940-1950 ● In the 1940s and 1950s, Zone electrophoresis ● A homogenous buffer system is
methods became effective which used filter paper used over the whole
or gels as supporting media. separation time and range to
ensure a constant pH value.
1960 ● By the 1960s, gel electrophoresis methods are ● Migration distances during a
introduced which can separate biological molecules defined time limit will measure
based on minute physical and chemical the electrophoretic mobilities of
differences. the substances.
○ Physical like molecular weight, molecular size ○ Through the defined time
and molecular shape, chemical differences (pH limit based on the guidelines
and charge). of the machine, we can be
● Used in several clinical and research laboratory able to identify the mobilities
testing: of the substance based on
1. Biochemistry - analysis of proteins, nucleic the distance of their
acids, etc. migration.
2. Immunology & Serology - antibodies and ● Can be applied to
antigens non-amphoteric and
3. Molecular biology and diagnostics - DNA amphoteric molecules.
analysis, DNA sequencing and etc. ● During separation, diffusion
4. Pharmaceuticals - vaccine testing, antibiotic can lead to blurred zones -

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reducing sensitivity of detection
and resolution.
● Buffer reservoirs at anodal
and cathodal sides are needed
to maintain buffer conditions
during separation.
ISOTACHOPHORESIS ● Separation is carried out in a
discontinuous buffer system
● Ionized sample migrates
between a leading electrolyte
with high mobility and a
terminating (aka trailing) ion with
low mobility, all migrating with
the same speed.
○ Your analyte/samples
will be in between the
leading and trailing
ions. None of them
will be in front of the
leading and none of
them will go later than
the trailing ion.
● Different components are
separated according to their
electrophoretic mobilities and
form stacks - the substance with
highest mobility follows
directly the leading ion; the
one with lowest mobility
migrates directly in front of
the terminating electrolyte.
ISOELECTRIC ● Takes place in a pH gradient.
FOCUSING (IEF) ● Can only be used for
amphoteric substances such
as peptides and proteins.
● Charged molecules move
toward the anode or the cathode
until they reach a position in the
pH gradient where their net
charges are zero.
● This pH value is the “isoelectric
point” of the substance.
● No problem with diffusion.
THE RATE OF MIGRATION OF A SOLUTE IN AN ELECTRIC
FIELD DEPENDS ON THE FOLLOWING FACTORS
1. Net charge on the particle
2. Size and Shape of the particles
3. pH of the medium
4. Strength of electric field
5. Chemical and Physical Properties of supporting medium
6. Electrophoretic Temperature
COMPONENTS OF ELECTROPHORESIS
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1. THE DRIVING FORCE (ELECTRICAL POWER)
or POWER SUPPLY
● Supplies constant current or voltage in the system
● Needed to mobilize particles through the support medium
● Also called the driving force
● The larger the separation distance, the higher the current
necessary to reach the specific field strength
● At a given ionic strength, the field strength is proportional to
the cross-section - the thicker the gel, the greater the current.
● The power is directly proportional to the volume of the gel
2. THE BUFFER SYSTEM
Buffer ions actually have a double purpose in electrophoresis:
1. They carry the applied current.
2. They set the pH at which electrophoresis is carried out
● Electrophoresis is always carried out in an appropriate buffer.
● It is essential to maintain a constant state of ionization of the
molecules being separated.
● The pH and ionic strength of buffer will affect the analyte
● Choice of buffer depends on the nature of substance to be
separated
● During electrophoresis, ions cluster around a migrating
particle.
● The higher the ionic concentration, the higher the size of the
ionic cloud and the lower the mobility of the particle.
● Greater ionic strength produces sharper protein-band
separation but leads to increased heat production.
● This may cause denaturation of heat-labile proteins.
Consequently, the optimal buffer concentration should be
determined for any electrophoretic system.
● Generally, the most widely used buffers are made of
monovalent ions because their ionic strength and molality are
equal.
Commonly used buffers for electrophoresis of DNA
TBE Tris borate EDTA
TPE Tris phosphate EDTA
TAE Tris acetate EDTA
3. THE SUPPORT MEDIUM
● A network of interacting fibers or a polymer that is solid but
traps large amounts of solvent in pores or channels inside.
● The support medium cuts down convection currents and
diffusion so that the separated components remain as sharp
zones.
FORMATS
Electrophoresis in Free a. Moving boundary
Solution electrophoresis
b. Free-flow electrophoresis
c. Capillary electrophoresis
Electrophoresis in a. Paper and thin-layer
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Supporting Media electrophoresis
b. Cellulose acetate
membrane electrophoresis
Gel Electrophoresis Gel Types
● Starch gels
● Dextran gels
● Agarose gels
● Polyacrylamide gels
GEL TYPES
Starch gel ● Introduced by Smithies (1955)
● Prepared from hydrolyzed potato starch -
dissolved by heating and poured to
thickness of 5-10 mm.
● Pore size adjustable by the starch
concentration in the solution.
Dextran gels ● Solely used for preparative methods
without sieving effect as in Isoelectric
Focusing (IEF)
Agarose gel ● Widely used supporting medium
obtained from red seaweed
● Used as a purified fraction of agar, it is
neutral and, therefore, does not
produce electroendosmosis
● Can separate proteins into 10-15 bands.
● Mostly used when needing large pores
for the analysis of molecules >10nm in
diameter.
● Gels of varying electroendosmosis and
degrees of purity can be obtained by the
removal of agaropectin.
● Pore size depends on concentration of
agarose (weight of agarose and volume
of water).
Polyacrilamid ● Involves separation of protein on the
e Gel basis of charge and molecular size.
● First used for electrophoresis by
Raymond and Weinstraub (1959).
● Layers of gel with different pore sizes
are used.
● Polyacrylamide gel electrophoresis
separates serum proteins into 20 or
more fractions rather than the usual 5
fractions separated by cellulose acetate
or agarose
● It is widely used to study individual
proteins (e.g. isoenzymes).
4. THE SAMPLE / ANALYTE
Without the sample or analyte, there would be no electrophoresis and
there is no separation of molecules.
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Mag-agad sila sa atong sample or analyte, for example, concentration of agar.
Mag-agad siya sa atong ganahan na iseparate nila nga molecules para didto ta
mag-agad saiyang pore size also the buffer used. Mag-agad sd ta saiyang sample
or analyte na iyang giprocess.
5. THE DETECTING MEDIUM
● Agents most frequently used for visualization of bands
○ Fluorescent dyes
○ Silver Stain
FLUORESCENT DYES
TYPES OF FLUORESCENT DYES
Intercalating Agents ● Intercalate or stack between the
nitrogen bases in double-stranded
nucleic acid.
● Most widely used - Ethidium
bromide (EtBr)
○ Under excitation with UV light at
300nm, EtBr in DNA will emit
orange light at 590 nm.
● After electrophoresis gel is soaked
in a solution of EtBr in running
buffer.
● Alternatively, dye is added directly to
the gel before polymerization or to
the running buffer.
● After soaking or running in dye, the
DNA illuminated with UV light will
appear as orange band in the gel.
Minor 1. SYBR green - most widely used set
Groove-Binding Dyes of stains
2. SYBR green I - for ds-DNA staining
3. SYBR green II - for ss-DNA or RNA
staining
4. SYBR gold - for both DNA and RNA
staining
● Along with similar methods with
EtBr, SYBR green can also be
added directly to the DNA sample
before electrophoresis.
● DNA prestaining decreases the
amount of dye required for DNA
visualization but lowers the
sensitivity of detection and may
interfere with DNA migration through
the gel at higher DNA
concentrations.
● Not mutagenic and is safe to use.
SILVER STAIN
● Silver diamine or Silver nitrate
● More sensitive than fluorescent dyes.
● Useful for protein analysis and detection of limiting
amounts of product.
● More complicated than simple fluorescent stains
● Color development must be carefully watched in order to
stop the reaction once optimal signal is reached.
● Overdevelopment of color reaction results to high
backgrounds and masking of results.
● Insoluble black silver salt precipitates upon introduction of
formaldehyde in a weak acid solution, or alkaline solution for
silver nitrate.
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OTHER DETECTING SYSTEMS
UV VISUALIZATION ● Directly placing the support
medium under a UV light.
● Simplest way to detect.
DENSITOMETER ● Device that measure the degree of
darkness (optical density) of a
photographic or semitransparent
material or of a reflecting surface.
TYPES OF ELECTROPHORESIS
ELECTROPHORESIS OF NUCLEIC ACIDS
● Each phosphate group on a nucleic acid polymer is
ionized, making the molecule negatively charged.
● Under an electric current, DNA and RNA will migrate toward
the positive pol (anode).
● In a matrix of agarose or polyacrylamide, migration under
the pull of current is impeded, depending on the size of the
molecules and the spaces in the gel matrix.
○ The migration depends on the size of the molecules
and the spaces in the gel matrix especially in
agarose or polyacrylamide gels.
● Because each nucleotide has one negative charge, the
charge-to-mass ratio of molecules of different sizes will
remain constant.
● DNA & RNA will migrate at speeds inversely related to the
size or length of the polymer.
○ The longer the strands gel, the slower the speed
becomes.
GEL ELECTROPHORESIS
● Gel matrix provide resistance to the movement of
molecules under the force of an electric current.
● Prevent diffusion and reduce convection currents so that
separated molecules form a defined group called “band”.
● The best matrix should be unaffected by electrophoresis,
simple to prepare, and amenable to modification.
● Examples of the mediums used in Gel Electrophoresis:
Agarose & Polyacrylamide
AGAROSE GEL ELECTROPHORESIS (AGE)
● Comes in hydrated form or powdered form.
● Hydrated form may be purchased in various concentrations,
buffers and sizes ready for use.
● Powdered form requires preparation in the lab before use:
suspension in buffer and heating, before pouring into a mold.
● Usually in a laboratory setting, we use the powdered form
because it is easier to store as compared to the hydrated form
and we won’t be bothered by the expiration that much.
● Concentration of agarose dictates the size of the spaces in the
gel.
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● Small DNA fragments (50-500bp) – more concentrated
agarose gels (2-3%)
● Larger fragments (2,000-50,000bp) – lower agarose
concentration (0.5-1%)
● Smaller DNA fragments require a more concentrated
agarose gel and if the fragments are large, the agarose gel
concentration should be lowered in this case.
Caution: High agarose concentration will impede migration, while very
low concentrations produce a weak gel that is easily broken.
PULSE-FIELD GEL ELECTROPHORESIS
● A modification of gel electrophoresis designed for very large
pieces of DNA (50,000-250,000+ bp).
● Used in genotyping and genetic fingerprinting because we are
dealing with very large DNA fragments.
● Pulses of current are applied to the gel in alternating
dimensions to enhance migration. Which is why it is called
pulsed-field gel because in Agarose Gel Electrophoresis the
current or voltage of light is constant in the field. Here, it is
applied in pulses.
● Simplest approach: Field-Inversion Gel Electrophoresis
(FIGE)
● Works by alternating the positive and negative electrodes
during electrophoresis.
● DNA goes periodically forward and backward.
● Requires temperature control and a switching mechanism.
● Requires gel box, electrode, gel configurations, and
appropriate electronic control to accommodate the switching of
electric fields.
○ It has the same principle as you are sifting. Wherein
you go back and forth in order to sift the little or
smaller fragments.
POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)
● Useful in resolving very small DNA fragment, single-stranded
DNA, RNA, and proteins.
● Polyacrylamide is a synthetic material, allowing precise
control of the polymer properties and higher resolution than
what can be achieved with agarose.
● Requires a catalyst to polymerize.
● Can be very thin (~50 μm), making gel preparation difficult.
CAPILLARY ELECTROPHORESIS
● Analyte is resolved in a thin glass (fused silica) capillary
(length: 30- 100cm; internal diameter: 25- 100μm).
● Fused silica is used because it is the most transparent
material allowing for passage of fluorescent light.
● Capillary has negative charge along its walls, generated by
dissociation of hydroxyl ions from the molecules of silicone
● Establishes an electroosmotic flow when current is
introduced along capillary length.
● Optimal separation requires use of proper buffer to ensure that
solute being separated is charged.
● Separates particles:
○ by size (small – fast migration; large – slow
migration) and
○ by charge (negative – fast migration; positive –
slow migration).
● Fluorescent labels – covalently attached to nucleic acid to be
separated.
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 ● pg to ng quantities of fluorescently labeled, denatured nucleic ● The right photo (Paternity testing) instead of measurement of
acid in buffer containing formamide are introduced to the base pairs, it usually use the patterns kung who the father is
capillary depending on the pattern.
● Capillary is held at a denaturing temperature (50-60 ̊C).
● Sample goes into the capillary through electrokinetic,
hydrostatic, or pneumatic injection.
○ Electrokinetic injection – used for nucleic acid
analysis
● A platinum electrode close to the end of the capillary
undergoes a transient high-positive charge to draw sample
to end of the capillary.
○ para naa tay push sa molecules to go through the
capillary
● When current is established with positive charge on opposite
end of capillary, nucleic acids migrate into and through the
capillary.

ADVANTAGES OF CAPILLARY ELECTROPHORESIS

● Since you are dealing with laser and light detection in capillary
● Increased sensitivity and immediate detection of the
electrophoresis, so the results would be in graphs
particles.
● Horizontal lines – increasing size
● Standards, controls, and test samples can be run through the
● Vertical lines – fluorescence units
capillary together with the use of multiple color-detection
● Kung asa siya mo peak ang line, mas daghan siya. Kay diba
systems.
fluorescence man atong labelling sa capillary electrophoresis
● Eliminates lane-to-lane variations (as used in gel
kato atong light detector and atong lazer would count the
electrophoresis).
number base on the fluorescence labelling. Fluorescence units
● Labor and runtime are greatly decreased as compared to GE,
kung asa siya taas diha nimo makit an kung unsa ka daghan
although instrumentation may be costly and detection
and then kung dako kaayo siya would go farther horizontally,
requires fluorescent labeling of samples.
as you can see in this results.
● Analytical software programs can automatically analyze
results gathered by detector in capillary electrophoresis
instrument.

RESULTS AND AUTOMATION

● For example sa atong left makita nato atong DNA analysis.

● M stands for molecular ladders. Basis of the measurement of
the base pairs.
● Measurements will depend on the analyte that we are using or
the sample.
○ For example, the analyte is 200-300 nanometer in
size, of course atong gamiton na molecular ladder
would be in the measurement of hundreds.
● Diba? Kay lain sad kaayo mag used tag measurement of
thousands unya mag bana-bana ra ta? Kung asa siya kung
naa ba siya tunga sa one thousand ug zero, so mag buot-buot
nalang ta?
● TAKE NOTE: kung unsa na analyte or sample gi use. For
example, kung thousands pud of course we will use the
measurement of thousands not in hundredths or ten
thousands. If hundred daghan rapud kaayo. If ten thousands
di pus ta ka specifically or accurate identify kung asa siya
dapit.

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