Electrophoresis (Midterms)

PINES CITY COLLEGES
COLLEGE OF MEDICAL LABORATORY SCIENCE

2nd SEMESTER A.Y. 2022-2023
MOLECULAR BIOLOGY LEC
TITLE: ELECTROPHORESIS (MIDTERM)
OBJECTIVES:
 Explain the principle and performance of electrophoresis as it applies to nucleic
acids.
 Compare and contrast the agarose and polyacrylamide gel polymers commonly used
to resolve nucleic acids, and state the utility of each polymer.
 Explain the principle and performance of capillary electrophoresis as it is applied
to nucleic acid separation.
 Describe the buffer used in electrophoresis.
ELECTROPHORESIS
 The movement of molecules by an _____________________.
 This can occur in solution, but it is practically done in a matrix to limit migration and
contain the migrating material.
 Routinely applied to the analysis of _______________________.
 Each phosphate group on a DNA polymer is ionized, making DNA a _______________
charged molecule.
 Under an electric current, DNA will migrate toward the __________________
 When DNA is applied to a macromolecular cage such as ________________________,
its migration under the pull of the current is impeded.
 Because each nucleotide has ___________________ charge, the charge-to-mass ratio
of molecules of different sizes will remain _________.
 DNA fragments will therefore migrate at speeds__________ related to their size.
 Can be performed in:
a) tubes
b) slab gels -either a horizontal or vertical format
c) capillaries.
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Gel Systems
 Gel matrices provide ____________ to the movement of molecules under the force of
the electric current.
 They prevent _______ and reduce ________ currents so that the separated molecules
form a defined group, or “band.”
 The gel can then serve as a ____________ for analysis of the separated components.
 Simple to prepare and amenable to modification.
Agarose Gels
 A polysaccharide polymer extracted from ________.
 It is a component of ____ used in bacterial culture dishes.
 A linear polymer of _________, which consists of 1,3-linked--D-galactopyranose and 1,4-
linked 3,6-anhydro--L-galactopyranose
 Agarose can be purchased and stored in the laboratory in powdered form.
 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
 Small pieces of DNA are resolved on __________________ agarose gels,
e.g., 2%–3%
 Larger fragments of DNA are best resolved in ____ agarose concentrations,
e.g., 0.5%–1%.
 Agarose concentrations above ___ and below ____ are not practical.
 High-concentration agarose will _____ migration
 Very low concentrations produce a _____ gel with limited integrity
Choice of Polyacrylamide Concentration for DNA Gels
Acrylamide Concentration (%) Separation Range (size in bp)
3.5 100-1000
5.0 80-500
8.0 60-400
12.0 40-200
20.0 10-100
Choice of Agarose Concentration for DNA Gels
Agarose Concentration (%) Separation Range (size in bp)
0.3 5000-60,000
0.6 1000-20,000
0.8 800-10,000
1.0 400-8000
1.2 300-7000
1.5 200-4000
2.0 100-3000
Pulsed Field Gel Electrophoresis

 Very ________ pieces of DNA cannot be resolved efficiently by simple agarose
electrophoresis.
Example: 50,000–250,000 bp
 Even in the ________ concentrations of agarose, megabase fragments are too severely
impeded for correct resolution.
 Limiting mobility is reached when a DNA molecule can move only lengthwise through
successive pores of the gel, called _________.
 For genomic-sized DNA molecules, pulses of current applied to the gel in alternating
dimensions enhance ____________ called __________________________________
(PFGE).
 The simplest approach to this method is _____________________________ (FIGE).
 FIGE works by alternating the positive and negative electrodes during
electrophoresis.
 The DNA goes periodically forward and backward.
 Requires _____________ control and a ___________ mechanism.
Examples of commonly used pulsed field or transverse angle reorientation
electrophoresis:
a) Contour-clamped homogeneous electric field
b) transverse alternating field electrophoresis
c) rotating gel electrophoresis
 Require a special ________ with a special __________ and ______________ as well as
appropriate electronic control for switching the electric fields during electrophoresis.
 Using PFGE, the ______ fragments are resolved, not only by sifting through the spaces
in the polymer but also by their reorientation and the time necessary to realign
themselves to move in a second dimension, usually an angle of 120 degree and 180 degree
for FIGE from the original direction of migration.
 DNA to be resolved by these methods must be protected from ___________________.
 Specimens are immobilized in an agarose plug before cell _____.
 Further treatment of the DNA, e.g., with restriction enzymes, is also performed while
the DNA is immobilized in the agarose plug. 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 _________________ that are set by the operator.
 These parameters are based on the general size of the fragments to be analyzed;
Example: a larger fragment will require a longer switch interval.
 PFGE is a ______ migration method. Sample runs will take ____ hours or more.
 _________________________ is used for applications that require the resolution of
chromosome-sized fragments of DNA such as in bacterial typing for epidemiological
purposes.
 Digestion of genomic DNA with restriction enzymes will yield a band pattern specific to
each type of organism.
 By comparing band patterns, the similarity of organisms isolated from various sources can
be assessed.
 This information is especially useful in determining the epidemiology of infectious
diseases
Example: identifying whether two biochemically identical isolates have a common source
PFGE FIGE
Polyacrylamide Gels
 Very ______ DNA fragments and ______________ DNA are best resolved on
polyacrylamide gels in polyacrylamide gel electrophoresis (PAGE).
 Acrylamide, in combination with the cross-linker methylene bisacrylamide _________
into a gel that has consistent resolution characteristics.
 Originally used mostly for ___________ separation, but it is now routinely applied to
_____________ analysis.
 USED FOR:
a) sequencing nucleic acids
b) mutation analyses
c) nuclease protection assays
 FORMS OF ACRYLAMIDE:
a) Powdered form is a dangerous __________ and must be handled with care.
b) Solutions of mixtures of acrylamide and bis-acrylamide are less hazardous and
more convenient to use.
c) Preformed gels are the most convenient
 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.
Example: 6% 19:1 acrylamide:bis gel has a T value of 6% and a C value of 5%.
 Unlike agarose gels that polymerize upon cooling, polymeration of polyacrylamide gels
requires the use of a __________.
 The catalyst may be the nucleation agents, ammonium persulfate (APS) plus
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 _______
plus TEMED.
 Deaeration, or the removal of air, of the gel solution is often done before the
addition of the nucleation agents.
 Polyacrylamide gels for nucleic acid separation are very thin
e.g., 50 um.
 The main advantage of polyacrylamide over agarose is the _______ resolution capability
for small fragments that can be accomplished with polyacrylamide.
ADVANTAGES:
a) A variation of 1 base pair in a 1-kb molecule (0.1% difference) can be detected in a
polyacrylamide gel.
b) Unlike agarose, the components of polyacrylamide gels are synthetic; thus, there is
not as much difference in batches obtained from different sources.
 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 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
 Applied to the separation of ___________________________
 An alternate method to ____ performance liquid chromatography (HPLC) for these
applications.
 Capillary electrophoresis has the advantage of faster analytical runs and lower cost per
run than HPLC.
 Used for the ________ and _________ of nucleic acids.
 In this type of electrophoresis, the analyte is resolved in a _________________
capillary that is 30–100 cm in length and has an internal diameter of 25–100 um.
 Fused silica is used as the capillary tube because it is the most ______________
material allowing for the passage of ______________.
 The fused silica is covered with a polyimide coating for protection. There is an
uncoated window where the light is shone on the fragments as they pass the
detector.
 The fused silica has a_________ 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.
 Capillary electrophoresis was originally applied to molecules in solution. Separation was
based on their size and charge (charge/mass ratio).
 Optimal separation requires the use of the proper _______ to ensure that the solute is
charged.
 Negatively charged molecules are completely ionized at _______,
 Positively charged solutes are completely protonated in _______buffers.
 Nucleic acids do not separate well in solution. As the size or length of a nucleic acid
increases so does its negative charge effectively confounding the charge/mass resolution.
 Introduction of a polymer inside the capillary restores resolution by retarding migration
according to size more than charge. It is important that the nucleic acid be completely
______________ so that it will be separated according to its size
 PROCEDURE:
a) 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.
b) The sample is injected into the capillary by:
1. Electrokinetic
2. Hydrostatic
3. pneumatic injection.
 For nucleic acid analysis, ______________ injection is used.
c) The platinum electrode close to the end of the capillary undergoes a transient high-
positive charge to draw the sample into the end of the capillary.
d) 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 to run the samples at higher charge per unit area,
which means that the samples migrate faster, thereby decreasing the resolution (run)
time.
 Nucleic acid resolution by capillary electrophoresis is used extensively in _________
applications and parentage
testing performed by analyzing short tandem repeat polymorphisms.
OTHER APPLICATIONS :
a) clonality testing
b) microsatellite instability detection
c) 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.
 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.
 Analytical software can automatically analyze results that are gathered by the detector
in the capillary electrophoresis instrument.
Buffer Systems
 Buffer system carry the______ and __________ during electrophoresis.
 A buffer is a solution of a _______ and its ___________.
 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
where [H+], [A- ], and [HA] represent the dissociated proton, dissociated base, and
associated salt concentrations, respectively. Ka is most commonly expressed as its
negative logarithm, pKa , such that
pKa= -log Ka
A pKa of 2 (Ka 10-2) favors the release of protons.

A pKa of 12 (Ka 10-12) favors the association of protons.
 A given buffer maintains the pH of a solution near its pKa. The amount the pH of a buffer
will differ from the pKa is expressed as the Henderson-Hasselbach equation:
 If the acidic and the basic forms of the buffer in solution are of equal concentration,
_______________ .
 If the acidic form predominates, the pH will be _____than the pKa
 if the basic form predominates, the pH will be______ than the pKa .
 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 of 1/10 that of the other form.
 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.
 The current through the gel is carried by buffer ions, preventing severe ____________ in
the pH of the gel.
 A buffer concentration must be _____ enough to provide sufficient acidic and basic forms
to buffer its solution.
 __________ the buffer concentration 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 ____________.
 High buffer concentrations must therefore be offset by _________.
 The gel system must be immersed in a buffer that conducts the electric current
efficiently in relation to the buffering capacity.
 Ions with high-charge differences, +2, -2, +3, ect move through the gel more quickly.This
results in too much current passing through the gel as well as faster depletion of the
buffer.
 Buffer components such as _______________________ are preferred because they
remain partly uncharged at the desired pH and thus maintain constant pH without high
conductivity.
 In addition to pKa, charge, and size, other buffer characteristics that can be taken into
account when choosing a buffer include toxicity, interaction with other components,
solubility, and ultraviolet absorption.
MOST COMMONLY USED FOR ELECTROPHORESIS:
a. Tris buffers Tris borate EDTA (TBE; 0.089 M Trisbase, 0.089 M boric acid, 0.0020 M
EDTA)
b. Tris phosphate EDTA (TPE; 0.089 M Tris-base, 1.3% phosphoric acid, 0.0020 M EDTA)
c. Tris acetate EDTA (TAE; 0.04 M Tris-base, 0.005 M sodium acetate, 0.002 M EDTA)
ADVANTAGES AND DISADVANTAGES OF BOTH TBE AND TAE
 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.
 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.
 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
 Used to modify sample molecules in ways that affect their migration.
Examples : formamide, urea, and various detergents.
 Denaturing agents, such as formamide or urea, break ________________ 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 _________________ the hydrogen
bonding sites, hindering complementary sequences from reannealing. As a result, the
molecules become long, straight, unpaired chains.
 Urea and heat in the gel systems maintain this conformation such that intrachain
_________________________ of the nucleic acid molecules does not affect migration
speeds, and separation can occur strictly according to the size or length of the molecule.
 Electrophoresis of RNA requires different conditions imparted by different additives
than are used with DNA. Because RNA is single-stranded and it tends to fold to optimize
internal homology, it must be completely ____________to prevent folding in order to
accurately determine its size by migration in a gel system.
 The secondary structures formed in RNA are strong and more difficult to denature than
DNA homologies.
 Denaturants used for RNA include ____________________ (MMH), which reacts with
amino groups on the RNA preventing base pairing between homologous nucleotides, and
aldehydes which also disrupt base pairing.
 RNA can be separated in 10-mM sodium phosphate, pH 7, or MOPS buffer (20 mM 3-[N-
morpholino] propanesulfonic acid, pH 7, 8-mM sodium acetate, 1 mM EDTA, pH 8).
 The RNA sample is incubated in dimethyl sulfoxide, 1.1 M glyoxal (ethane 1.2 dione) and
0.01 M sodium phosphate, pH 7, to denature the RNA prior to loading the sample on the
gel.
 Due to pH drift during the run, the buffer should be recirculated from the anode end of
the bath to the cathode end This can be accomplished using a peristaltic pump or by
stopping the gel at intervals and transferring the buffer from the cathode to the anode
ends.
References:
 Molecular Cell Biology Ninth Editio ©2021 Harvey Lodish; Arnold Berk; Chris A. Kaiser;
Monty Krieger; Anthony Bretscher; Hidde Ploegh; Kelsey C. Martin; Michael Yaffe;
Angelika Amon
 Molecular Biology Made Simple and Fun, Third Edition 3rd Edition by David P. Clark
Education is the passport to the future, for tomorrow belongs to those who prepare for it
today.
-Malcolm X-

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PINES CITY COLLEGES

COLLEGE OF MEDICAL LABORATORY SCIENCE

TITLE: ELECTROPHORESIS (MIDTERM)

Pulsed Field Gel Electrophoresis

A pKa of 2 (Ka 10-2) favors the release of protons.

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