Resolution and

Detection of
Nucleic Acids
SABAHAT AZIZ
SENIOR LECTURER
CLINICAL LABORATORY SCIENCES
INSTITUTE OF MEDICAL TECHNOLOGY
DOW UNIVERSITY OF HEALTH
SCIENCES

DIMT- Advance clinical pathology III MT(CLS)-606 18th Sep 2023 1

 LECTURE OUTLINE

ELECTROPHORESIS GEL SYSTEMS

• Agarose Gels
• Polyacrylamide Gels
• Capillary Electrophoresis
BUFFER SYSTEMS
• Buffer Additives
ELECTROPHORESIS EQUIPMENT GEL LOADING
DETECTION SYSTEMS
• Nucleic Acid–Specific Dyes
• Silver Stain
 At the end of class students will be able to know

• MEASUREMENT OF NUCLEIC ACID QUALITY AND QUANTITY

Electrophoresis, Spectrophotometry, Fluorometry

DIMT- Advance clinical pathology III MT(CLS)-606 18th Sep 2023 3

 How gel electrophoresis is useful? And
what factors Influences?​
It helps in the separation of individual DNA, RNA, proteins from its
mixture.​

Mixtures of molecules separates based on their movement.

Separation depends on:​

▪ Charge on the molecule ​- helps in movement of molecule in

electric field

▪ Size of molecule – differential separation

 A General Gel Electrophoresis Set-up​
▪ Gel (solid matrix support)​

▪ Electric field (supported with negative & positive

poles)

▪ Buffer (liquid medium) ​

DIMT- Advance clinical pathology III MT(CLS)-606 18th Sep 2023 6
 Electrophoresis

• Electrophoresis is the movement of molecules by an electric current.

• This can occur in solution, but it is practically done in a matrix to limit
migration and contain the migrating material.
• Electrophoresis is routinely applied to the analysis of proteins and nucleic
acids.
• Under an electric current, DNA will migrate toward the positive pole (anode).
• When DNA is applied to a macromolecular cage such as agarose or
polyacrylamide, its migration under the pull of the current is impeded,
depending on the size of the DNA and the spaces in the gel.
DIMT- Advance clinical pathology III MT(CLS)-606 18th Sep 2023 8
 ELECTROPHORESIS

DNA fragments will therefore migrate at speeds inversely related to their

size.
• Electrophoresis can be performed in slab gels, or capillaries.

• Slab gel electrophoresis can have either a horizontal or vertical format

(Fig.1).
 GEL SYSTEMS

• Gel matrices provide resistance to the movement of molecules under the force
of the electric current.
• They prevent diffusion and reduce convection currents so that the separated
molecules form a defined group, or “band.”
• The gel can then serve as a support medium for analysis of the separated
components.
• These matrices must be simple to prepare .
• Agarose and polyacrylamide are polymers that meet these criteria.
 AGAROSE GELS

• Agarose is a polysaccharide polymer extracted from seaweed.

• It is a component of agar used in bacterial culture dishes.
• Agarose is a linear polymer of agarobiose, which consists of 1,3-linked--D-
galactopyranose and 1,4- linked 3,6-anhydro--L-galactopyranose (Fig.2).
• Hydrated agarose gels in various concentrations, buffers, and sizes can be purchased
ready for use.
• Alternatively, agarose can be purchased and stored in the laboratory in powdered
form.
• For use, powdered agarose is suspended in buffer, heated, and poured into a
mold.
• The concentration of the agarose dictates the size of the spaces in the gel and will,
therefore, be determined by the size of DNA to be resolved (Table 1).
 AGAROSE GELS

• Agarose is a polysaccharide polymer extracted from seaweed.

• It is a component of agar used in bacterial culture dishes.
• Agarose is a linear polymer of agarobiose, which consists of 1,3-linked--D-
galactopyranose and 1,4- linked 3,6-anhydro--L-galactopyranose (Fig.2).
• Alternatively, agarose can be purchased and stored in the laboratory in
powdered form.
• For use, powdered agarose is suspended in buffer, heated, and poured
into a mold.
• The concentration of the agarose dictates the size of the spaces in the gel
and will, therefore, be determined by the size of DNA to be resolved (Table
1).
 1% refers to the percentage of
agarose in the volume of liquid. The
gel percentage is calculated as
(grams of agarose / milliliters of
buffer) x 100%. In this gel, we are
mixing 0.5g with 50mL, so the
calculation is 0.5g / 50 mL x 100%,
which gives us a 1% gel.

1 g of agarose powder dissolved in 100 ml buffer yields a 1% gel.

2 g of agarose in 100 mL of TAE will make a 2% gel)

 AGAROSE GELS

• Small pieces of DNA (50–500 bp) are resolved on more concentrated agarose gels,
e.g., 2%–3% (Fig.3).
• Larger fragments of DNA (2000–50,000) are best resolved in lower agarose
concentrations, e.g., 0.5%–1%.
• Agarose concentrations above 5% and below 0.5% are not practical.
• High-concentration agarose will impede migration, whereas very low
concentrations produce a weak gel with limited integrity.
 Agarose gel electrophoresis: Procedure
Step 1: Preparation of Agarose Gel

▪ Agarose (to prepare gel/solid matrix)

▪ TAE Buffer concentrated (to maintain
appropriate ionic environment
supporting the movement of charged
molecules)
▪ Deionized water (to dilute buffer)
▪ Gel casting tray
▪ Comb to create wells for sample
 Agarose gel electrophoresis: Procedure
Step 2: Running the Agarose
Gel
▪ Sample loading dye
▪ 1X TAE Buffer (to maintain
appropriate ionic environment
supporting the movement of
charged molecules)
▪ Deionized water (to dilute buffer)
▪ Electrophoresis tank & power
supply
 PULSED FIELD GEL ELECTROPHORESIS

• Very large, i.e., 50,000–250,000 bp, pieces of DNA cannot be resolved

efficiently by simple agarose electrophoresis.
• Even in the lowest concentrations of agarose, megabase fragments are
too severely impeded for correct resolution (referred to as limiting
mobility).
• Limiting mobility is reached when a DNA molecule can move only
lengthwise through successive pores of the gel, a process called
reptation.
• For genomic-sized DNA molecules, pulses of current applied to the gel in
alternating dimensions enhance migration.
• This process is called pulsed field gel electrophoresis (PFGE) (Fig.4).
 PULSED FIELD GEL ELECTROPHORESIS

• The simplest approach to this method is field inversion gel electrophoresis (FIGE).
• FIGE works by alternating the positive and negative electrodes during electrophoresis.
• In this type of separation, the DNA goes periodically forward and backward.
• This method requires temperature control and a switching mechanism.
• Contour-clamped homogeneous electric field, transverse alternating field electrophoresis, and
rotating gel electrophoresis are examples of commonly used pulsed field or transverse angle
reorientation electrophoresis.
• These systems require a special gel box with a special electrode and gel configuration as well as
appropriate electronic control for switching the electric fields during electrophoresis
 PULSED FIELD GEL ELECTROPHORESIS

• Using PFGE, the large fragments are resolved,

• DNA to be resolved by these methods must be protected from
breakage and shearing.
• Therefore, specimens are immobilized in an agarose plug before cell
lysis.
• Further treatment of the DNA, e.g., with restriction enzymes, is also
performed while the DNA is immobilized in the agarose plug.
 PULSED FIELD GEL ELECTROPHORESIS

• After treatment, the plug is inserted directly into the agarose gel for
electrophoresis.
• PFGE instruments are designed to apply current in alternating directions at
specific times (called the switch interval) that are set by the operator.
• These parameters are based on the general size of the fragments to be
analyzed; i.e., a larger fragment will require a longer switch interval.
• PFGE is a slow migration method.
• Sample runs will take 24 hours or more.
• Alternating field electrophoresis is used for applications that require the
resolution of chromosome-sized fragments of DNA such as in bacterial typing
for epidemiological purposes
 POLYACRYLAMIDE GELS

• Very small DNA fragments and single-stranded DNA are best resolved on
polyacrylamide gels in polyacrylamide gel electrophoresis (PAGE).
• Acrylamide, in combination with the cross-linker methylene bisacrylamide
(Fig.5), polymerizes into a gel that has consistent resolution characteristics
(Fig.6).
• Polyacrylamide was originally used mostly for protein separation, but it is
now routinely applied to nucleic acid analysis.
• Polyacrylamide gels are used for sequencing nucleic acids, mutation analyses,
nuclease protection assays, and other applications requiring the resolution of
nucleic acids down to the single-base level.
• Acrylamide is supplied to the laboratory in several forms.
DIMT- Advance clinical pathology III MT(CLS)-606 18th Sep 2023 27
Polyacrylamide Gel electrophoresis (PAGE): Procedure

Stacking Vs Resolving gel

 Polyacrylamide Gel
electrophoresis (PAGE)

Step 3: Sample loading &

Running the Gel
Polyacrylamide Gel
electrophoresis
(PAGE): Quick
Procedure
 POLYACRYLAMIDE GELS
• The powdered form is a dangerous neurotoxin and must be handled with
care.
• Solutions of mixtures of acrylamide and bis-acrylamide are less hazardous
and more convenient to use.
• Preformed gels are the most convenient, as the procedure for preparation of
acrylamide gels is more involved than that for agarose gels.
• The composition of polyacrylamide gels is represented as the total
percentage concentration (w/v) of monomer (acrylamide with cross-linker) T
and the percentage of monomer that is cross-linker C.
• For example, a 6% 19:1 acrylamide:bis gel has a T value of 6% and a C
value of 5%
 POLYACRYLAMIDE GELS

• Unlike agarose gels that polymerize upon cooling, polymeration of

polyacrylamide gels requires the use of a catalyst.
• The catalyst may be the nucleation agents, ammonium persulfate (APS) plus
N,N,N,N-tetramethylethylenediamine (TEMED), or light activation.
• APS produces free oxygen radicals in the presence of TEMED to drive the
free-radical polymerization mechanism.
• Free radicals can also be generated by a photochemical process using
riboflavin plus TEMED.
• Excess oxygen inhibits the polymerization process. Therefore, deaeration,
or the removal of air, of the gel solution is often done before the addition of
the nucleation agents.
 POLYACRYLAMIDE GELS

• Polyacrylamide gels for nucleic acid separation are very thin, e.g., 50 m,
making gel preparation difficult.
• Systems have been designed to facilitate the preparation of single and
multiple gels. Increasing numbers of laboratories are using preformed
polyacrylamide gels to avoid the hazards of working with acrylamide
and the labor time involved in gel preparation.
• Use of preformed gels must be scheduled, keeping in mind the limited
shelf life of the product.
• The main advantage of polyacrylamide over agarose is the higher
resolution capability for small fragments that can be accomplished with
polyacrylamide.
 POLYACRYLAMIDE GELS

• A variation of 1 base pair in a 1-kb molecule (0.1% difference) can be detected in a

polyacrylamide gel.
• Another advantage of polyacrylamide is that, unlike agarose, the components of
polyacrylamide gels are synthetic; thus, there is not as much difference in batches
obtained from different sources.
• Further, altering T and C in a polyacrylamide gel can change the pore size and,
therefore, the sieving properties in a predictable and reproducible manner.
• Increasing T decreases the pore size proportionally.
• The minimum pore size (highest resolution for small molecules) occurs at a C value
of 5%. Variation of C above or below 5% will increase pore size.
• Usually, C is set at 3.3% (29:1) for native and 5% (19:1) for standard DNA and
RNA gels.
 CAPILLARY ELECTROPHORESIS

• The widest application of capillary electrophoresis has been in the separation

of organic chemicals such as pharmaceuticals and carbohydrates.
• It has also been applied to the separation of inorganic anions and metal ions.
• It is an alternate method to high performance liquid chromatography (HPLC)
for these applications.
• Capillary electrophoresis has the advantage of faster analytical runs and lower
cost per run than HPLC.
• Increasingly, capillary electrophoresis is being used for the separation and
analysis of nucleic acids, which is explained below.
• In this type of electrophoresis, the analyte is resolved in a thin glass (fused
silica) capillary that is 30–100 cm in length
 CAPILLARY ELECTROPHORESIS

• Fused silica is used as the capillary tube because it is the most transparent
material allowing for the passage of fluorescent light.
• The fused silica is covered with a polyimide coating for protection.
• The fused silica has a negative charge along the walls of the capillary
generated by the dissociation of hydroxyl ions from the molecules of
silicone.
• This establishes an electro-osmotic flow when a current is introduced along
the length of the capillary.
• Under the force of the current, small and negatively charged molecules
migrate faster than large and positively charged molecules (Fig.7).
• Capillary electrophoresis was originally applied to molecules in solution
 CAPILLARY ELECTROPHORESIS
• Separation was based on their size and charge (charge/mass ratio).
• Optimal separation requires the use of the proper buffer to ensure that the
solute is charged.
• Negatively charged molecules are completely ionized at high pH, whereas
positively charged solutes are completely protonated in low pH buffers.
• Nucleic acids do not separate well in solution.
• As the size or length of a nucleic acid increases (retarding migration), so does
its negative charge (speeding migration), effectively confounding the
charge/mass resolution.
• Introduction of a polymer inside the capillary restores resolution by retarding
migration according to size more than charge.
 CAPILLARY ELECTROPHORESIS
• It is important that the nucleic acid be completely denatured (single-
stranded) so that it will be separated according to its size, because secondary
structure will affect the migration speed.
• Generally, 1–50 nL of denatured nucleic acid in buffer containing
formamide is introduced to the capillary, which is held at a denaturing
temperature through the run.
• The sample is injected into the capillary by electrokinetic, hydrostatic, or
pneumatic injection.
• For nucleic acid analysis, electrokinetic injection is used.
• Electrokinetic injection takes place when a voltage is applied to the electrode
inside the sample solution driving the analytes to the capillary.
 CAPILLARY ELECTROPHORESIS
• When the current is established, the fragments migrate through the
capillary.
• For the resolution of nucleic acids, capillary electrophoresis is
analogous to gel electrophoresis with regard to the electrophoretic
parameters.
• The capillary’s small volume, as compared with that of a slab gel, can
dissipate heat more efficiently during the electrophoresis process.
• More efficient heat dissipation allows the technologist to run the
samples at higher charge per unit area, which means that the samples
migrate faster, thereby decreasing the resolution (run) time.
 CAPILLARY ELECTROPHORESIS
• Nucleic acid resolution by capillary electrophoresis is used extensively in forensic
applications and parentage testing performed by analyzing short tandem repeat
polymorphisms.
• It has other applications in the clinical laboratory, such as clonality testing,
microsatellite instability detection, and bone marrow engraftment analysis.
• Specially designed software can use differentially labeled molecular weight
markers or allelic markers that, when run through the capillary with the sample,
help to identify sample bands.
 CAPILLARY ELECTROPHORESIS
• The capillary system has the advantages over traditional slab gel
electrophoresis of increased sensitivity, so that smaller amounts of nucleic
acid can be analyzed, and immediate detection of desired bands.
• With multiple color detection systems, standards, controls, and test samples
can be run through the capillary together, thereby eliminating the lane-to-
lane variations that can occur across a gel.
• Although instrumentation for capillary electrophoresis is costly and detection
requires fluorescent labeling of samples that can also be expensive, labor
and run time are greatly decreased as compared with gel electrophoresis.
• In addition, analytical software can automatically analyze results that are
gathered by the detector in the capillary electrophoresis instrument
 BUFFER SYSTEMS
• The purpose of a buffer system is to carry the current and protect the samples
during electrophoresis.
• This is accomplished through the electrochemical characteristics of the buffer
components.
• A buffer is a solution of a weak acid and its conjugate base.
• The pH of a buffered solution remains constant as the buffer molecules take up
or release protons given off or absorbed by other solutes.
• The equilibrium between acid and base in a buffer is expressed as the dissociation
constant, Ka:
 BUFFER SYSTEMS
• The Henderson-Hasselbach equation predicts that, in order to change the pH of a
buffered solution by one point, either the acidic or basic form of the buffer must be
brought to a concentration
• Therefore, addition of acid or base will barely affect the pH of a buffered solution
as long as the acidic or basic forms of the buffer are not depleted.
• Control of the pH of a gel by the buffer also protects the sample molecules from
damage.
• Furthermore, the current through the gel is carried by buffer ions, preventing
severe fluctuations in the pH of the gel
 BUFFER SYSTEMS
• A buffer concentration must be high enough to provide sufficient acidic
and basic forms to buffer its solution.
• Raising the buffer concentration, however, also increases the
conductivity of the electrophoresis system, generating more heat at a
given voltage.
• This can cause problems with gel stability and can increase sample
denaturation.
• High buffer concentrations must therefore be offset by low voltage.
• In order for nucleic acids to migrate properly, the gel system must be
immersed in a buffer that conducts the electric current efficiently in
relation to the buffering capacity
 BUFFER SYSTEMS
• The Tris buffers Tris borate EDTA (TBE; 0.089 M Trisbase, 0.089 M boric
acid, 0.0020 M EDTA), Tris phosphate EDTA (TPE; 0.089 M Tris-base,
1.3% phosphoric acid, 0.0020 M EDTA) and Tris acetate EDTA (TAE;
0.04 M Tris-base, 0.005 M sodium acetate, 0.002 M EDTA) are most
commonly used for electrophoresis of DNA.
• There are some advantages and disadvantages of both TBE and TAE that
must be considered before one of these buffers is used for a particular
application.
• TBE has a greater buffering capacity than TAE. Although the ion species
in TAE are more easily exhausted during extended or high-voltage
electrophoresis, DNA will migrate twice as fast in TAE than in TBE in a
constant current.
 BUFFER SYSTEMS
• TBE is not recommended for some post-electrophoretic isolation procedures.
• When using any buffer, especially TBE and TPE, care must be taken that the gel
does not overheat when run at high voltage in a closed container.
• Finally, stock solutions of TBE are prone to precipitation.
• This can result in differences in concentration between the buffer in the gel and the
running buffer.
• Such a gradient will cause localized distortions in nucleic acid migration patterns,
often causing a salt wave that is visible as a sharp horizontal band through the gel.
 BUFFER ADDITIVES

• Buffer additives are often used to modify sample molecules in ways that
affect their migration.
• Examples of these additives are formamide, urea, and various detergents.
• Denaturing agents, such as formamide or urea, break hydrogen bonds
between complementary strands or within the same strand of DNA or RNA.
• The conformation or solubility of molecules can be standardized by the
addition of one or both of these agents.
• Formamide and heat added to DNA and RNA break and block the hydrogen
bonding sites, hindering complementary sequences from reannealing.
• As a result, the molecules become long, straight, unpaired chains
 ELECTROPHORESIS EQUIPMENT
• Gel electrophoresis can be done in one of two conformations, horizontal or
vertical.
• In general, agarose gels are run horizontally, and polyacrylamide gels are run
vertically.
• Horizontal gels are run in acrylic containers called gel boxes or baths that are
divided into two parts with a platform in the middle on which the gel rests (Fig.9).
• Platinum wires make up the electrodes in the gel compartments.
• The wires are connected to a power source by banana clips or connectors through
the walls of the container.
• The gel in the box is submerged with electrophoresis buffer filling both
compartments and making a continuous system through which the current flows.
 ELECTROPHORESIS EQUIPMENT
• The thickness of the gel and the volume of the buffer affect migration, so
these parameters should be kept constant for consistent results.
• As the gel is submerged through the loading and electrophoresis process,
horizontal gels are sometimes referred to as submarine gels.
• The power supply will deliver voltage, setting up a current that will run
through the gel buffer and the gel, carrying the charged sample through the
matrix of the gel at a speed corresponding to the charge/mass ratio of the
sample molecules.
• Horizontal agarose gels are cast as square or rectangular slabs of varying size.
• Purchased gel boxes come with casting trays that mold the gel to the
appropriate size for the gel box.
 ELECTROPHORESIS EQUIPMENT
• The volume of the gel solution will deter-mine the thickness of the gel.
• Agarose, supplied as a dry powder, is mixed at a certain percentage
(w/v) with electrophoresis buffer and heated on a heat block or by
microwave to dissolve and melt the agarose.
• The molten agarose is cooled to 55–65C, and a certain volume is
poured into the casting tray as dictated by the gel box manufacturer or
application.
• A comb is then inserted into the top of the gel to create holes, or wells,
in the gel into which the sample will be loaded
 ELECTROPHORESIS EQUIPMENT
• The size of the teeth in the comb will determine the volume of loaded
sample and the number of teeth will determine the number of wells that
are available in the gel to receive samples.
• The gel is then allowed to cool, during which time it will solidify.
• After the gel has polymerized, the comb is carefully removed and the
gel is placed into the gel box and submerged in electrophoresis buffer.
• Vertical gel boxes have separate chambers that are connected by the gel
itself.
• Electrodes are attached to the upper and lower buffer chambers to set
up the current that will run through the gel
 ELECTROPHORESIS EQUIPMENT

• Vertical gel systems can range from large sequencing systems (35 cm x 26 cm)
to mini-systems (8 cm x 10 cm).
• Some mini-systems are big enough to accommodate two gels at a time (Fig.10).
• Mini-systems are used extensively for analyses that do not require single base
pair resolution.
• The larger systems are used for sequencing or other procedures requiring single-
base resolution.
• The gels are loaded from the top, below a layer of buffer in the upper chamber.
• Long, narrow gel-loading pipette tips that deposit the sample neatly on the floor
of the well increase band resolution and sample recovery.
 ELECTROPHORESIS EQUIPMENT
• The gel must be in place before filling the upper chamber with buffer.
• Some systems have a metal plate attached to the back of the gel to maintain
constant temperature across the gel.
• Maintaining constant temperature throughout the gel is more of a problem
with vertical gels because the outer edges of the gel cool more than the
center, slowing migration in the outer lanes compared with lanes in the
center of the gel.
• This is called “gel smiling” because similar-sized bands in the cooler outer
lanes will migrate slower than comparable bands in the inside lanes.
• Ensuring that there is no variation in temperature across the gel prevents gel
smiling from occurring.
 ELECTROPHORESIS EQUIPMENT

• Vertical gels are cast between glass plates that are separated by spacers.
• The spacers determine the thickness of the gel, ranging 0.05–4 mm.
• The bottom of the gel is secured by tape or by a gasket in specially designed gel casting
trays.
• After addition of polymerization agents, the liquid acrylamide is poured or forced between
the glass plates with a pipet or a syringe.
• The comb is then placed on the top of the gel.
• During this process, it is important not to introduce air into the gel or beneath the comb.
• Bubbles will form discontinuities in the gel, and oxygen will inhibit the polymerization of
the acrylamide.
• The comb is of a thickness equal to that of the spacers so that
• the gel will be the same thickness throughout.
 ELECTROPHORESIS EQUIPMENT

• As with horizontal gels, the number and size of the comb teeth determine the number
of wells in the gel and the sample volume that can be added to each well.
• Specialized combs, called shark’s-tooth combs, are often used for sequencing gels
(Fig.11).
• These combs are placed upside down (teeth up, not in contact with the gel) to form a
trough on the gel during polymerization.
• After polymerization is complete, the comb is removed and placed tooth-side down
on top of the gel for loading.
• With this configuration, the spaces between the comb teeth form the wells as opposed
to the teeth themselves forming the wells in the horizontal gels.
• The advantage to this arrangement is that the lanes are placed immediately adjacent
to one another to facilitate lane-to-lane comparisons.
 ELECTROPHORESIS EQUIPMENT

• When used, standard combs are removed before the gel is loaded, whereas the
shark’s-tooth combs are made so that the wells can be loaded while the comb is in
place.
• When the standard combs are removed from the gel, care must be taken not to
break or displace the “ears” that were formed by the spaces between the teeth in the
comb that separate the gel wells.
• Polyacrylamide gels can also be cast in tubes for isoelectric focusing or two-
dimensional gel electrophoresis.
• The tubes containing the gels are placed into a chamber separated as for vertical
slab gels.
• The tubes are held in place by gaskets in the upper chamber. This gel configuration,
however, limits the number of samples, as only one sample can be run per gel.
 GEL LOADING

• Prior to loading the sample containing isolated nucleic acid onto the gel,
tracking dye and a density agent are added to the sample.
• The density agent (either Ficoll, sucrose, or glycerol) increases the density
of the solution as compared with the electrophoresis buffer.
• When the sample solution is dispensed into the wells of the gel below the
surface of the buffer, it sinks into the well instead of floating away in the
buffer.
• The tracking dyes are used to monitor the progress of the
electrophoresis run.
• The dyes migrate at specific speeds in a given gel concentration and
usually run ahead of the smallest fragments of DNA
 GEL LOADING

• They are not associated with the sample DNA, and thus they do not affect the
separation of the sample DNA.
• The movement of the tracking dye is monitored, and when the tracking dye
approaches the end of the well electrophoresis is terminated.
• Bromophenol blue is a tracking dye that is used for many applications.
• Xylene cyanol green is another example of chromophores that are used as tracking
dyes for both agarose and polyacrylamide gels.
 Agarose gel electrophoresis: Procedure
Loading the sample: Use of sample loading dye
Components of loading dye:
- Xylene cyanol: travel with shorter fragments
- Bromophenol blue: travel with large fragments Mix loading dye and sample (1:5 ratio) and load in the well
- Glycerol: forms viscosity
 DETECTION SYSTEMS

• Following are the status of samples during and after electrophoresis is

accomplished using dyes that specifically associate with nucleic acid.
• The agents used most frequently for this application are fluorescent dyes and silver
stain.
 NUCLEIC ACID–SPECIFIC DYES

• Intercalating agents intercalate, or stack, between the nitrogen bases in double-

stranded nucleic acid.
• Ethidium bromide, 3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide (EtBr),
is one of these agents.
• Under excitation with ultraviolet light at 300 nm, EtBr in DNA emits visible light at
590 nm.
• Therefore, DNA separated in agarose or acrylamide and exposed to EtBR will emit
orange light when illuminated by ultraviolet light at 300 nm.
• EtBr was the most widely used dye in early DNA and RNA analyses.
• Care must be taken in handling EtBr because it is carcinogenic.
• After electrophoresis, the agarose or acrylamide gel can be soaked in a solution of
0.1–1–mg/ml EtBr in running buffer (TAE, TBE, or TPE) or TE.
White bands on dark gel due to staining with
ethidium bromide

DIMT- Advance clinical pathology III MT(CLS)-606 18th Sep 2023 66

 NUCLEIC ACID–SPECIFIC DYES
• Alternatively, dye can be added directly to the gel before polymerization or to the
running buffer.
• The latter two measures save time and allow visualization of the DNA during the run.
• Dye added to the gel, however, may form a bright front across the gel that could
mask informative bands.
• Dye added to the running buffer produces more consistent staining, although more
hazardous waste is generated by this method.
• Some enclosed gel systems contain EtBr inside a plastic enclosed gel cassette, limiting
exposure and waste.
• After soaking or running in EtBr, the DNA illuminated with ultraviolet light will appear
as orange bands in the gel.
• The image can be captured with a camera or by digital transfer to analytical software.
 NUCLEIC ACID–SPECIFIC DYES

• SyBr green is one of a set of stains introduced in 1995 as another type of nucleic
acid–specific dye system.
• It differs from EtBr in that it does not intercalate between bases; it sits in the minor
groove of the double helix.
• SyBr green in association with DNA or RNA also emits light in the orange range.
SyBr green staining is 25–100 times more sensitive than EtBr (detection level: 60
pg of double-stranded DNA vs. 5 ng for EtBr).
• This is due, in part, to background fluorescence from EtBr in agarose.
• A 1 x dilution of the manufacturer’s 10,000X stock solution of SyBr green in TAE,
TBE, or TE can be used in methods described for EtBr.
• A 1/100 dilution of SyBr green can also be added directly to the DNA sample before
electrophoresis.
 NUCLEIC ACID–SPECIFIC DYES
• Because SyBr green is not an intercalating agent, it is not as
mutagenic.
• Although SyBr green has some advantages over EtBr, many
laboratories continue to use the latter dye due to the requirement for
special optical filters for detection of SyBr green.
• Scanning and photographic equipment optimized for EtBr would have
to be modified for optimal detection of the SyBr green stains.
 NUCLEIC ACID–SPECIFIC DYES
• New instrumentation with more flexible detection systems allows
utilization of the SyBr green stains.
• SyBr green is the preferred dye for real-time PCR methods.
 SILVER STAIN

• A more sensitive staining system originally developed for protein visualization

is silver stain.
• After electrophoresis, the sample is fixed with methanol and acetic acid.
• The gel is then impregnated with ammoniacal silver (silver diamine) solutions
or silver nitrate in a weakly acid solution.
• Interaction of silver ions with acidic or nucleophilic groups on the target results
in crystallization or deposition of metallic silver under optimal pH conditions.
• The insoluble black silver salt precipitates upon introduction of formaldehyde
in a weak acid solution or alkaline solution for sliver nitrate.
• Of the two procedures, silver diamine is best for thick gels, whereas silver
nitrate is considered to be more stable
 SILVER STAIN
• Silver staining avoids the hazards of the intercalators, but silver nitrate is itself
also a biohazard.
• In addition, silver staining is more complicated than simple intercalation.
• Color development must be carefully watched as the precipitate accumulates in
order to stop the reaction once optimal signal is reached.
• Overexposure of the gel will result in high backgrounds and masking of
results.
• The increased sensitivity of this staining procedure, however, makes up for its
limitations.
• It is especially useful for protein analysis and for detection of limiting
amounts of product.
 Agarose gel electrophoresis: Documenting a Gel
▪ Expose the gel into ultraviolet light under gel documentation system (Modern method) /
UV transilluminator (conventional method)

UV transilluminator Gel Documentation System

 THANK YOU
sabahat.aziz@duhs.edu.pk

DIMT- Advance clinical pathology III MT(CLS)-606 18th Sep 2023 74