Resolution and Detection of
Nucleic Acids
S. Karutha Pandian
Department of Biotechnology
Alagappa University
Karaikudi – 630 003
www.alagappabiotech.org
Gel Electrophoresis
Electrophoresis is the movement of
molecules by an electric current.
Nucleic acid moves from a negative to a
positive pole.
Nucleic acid has a net negative charge,
they RUN TO RED
Electrophoresis of Nucleic Acids
Nucleic acids are separated based on size and
charge.
DNA molecules migrate in an electrical field at a
rate that is inversely proportional to the log10 of
molecular size (number of base pairs).
Employs a sieve-like matrix (agarose or
polyacrylamide) and an electrical field.
DNA possesses a net negative charge and
migrates towards the positively charged
anode.
Applications of Electrophoretic
Techniques in the Molecular Diagnostics
Laboratory
Sizing of Nucleic Acid Molecules
DNA fragments for Southern transfer analysis
RNA molecules for Northern transfer analysis
Analytical separation of PCR products
Detection of Mutations or Sequence
Variations
Principles of Gel Electrophoresis
Electrophoresis is a technique used to separate
and sometimes purify macromolecules
Proteins and nucleic acids that differ in size, charge or
conformation
Charged molecules placed in an electric field
migrate toward either the positive (anode) or
negative (cathode) pole according to their
charge
Proteins and nucleic acids are electrophoresed
within a matrix or "gel"
ELECTROPHORESIS
DNA and RNA are
negatively charged;
they RUN TO RED!
Principles of Gel Electrophoresis
The gel itself is composed of either
agarose or polyacrylamide.
Agarose is a polysaccharide extracted
from seaweed.
Polyacrylamide is a cross-linked polymer
of acrylamide.
Acrylamide is a potent neurotoxin and should
be handled with care!
Gel Electrophoresis Matrices
Agarose
Acrylamide
Types Of Nucleic Acid
Electrophoresis
Agarose gel electrophoresis
DNA or RNA separation
TAE or TBE buffers for DNA, MOPS with
formaldehyde for RNA
Polyacrylamide gel electrophoresis
(PAGE)
Non-denaturing (Special applications in
research)
Denaturing contain 6-7 M Urea (Most common)
Agarose Gel Electrophoresis
Separates fragments based on mass,
charge
Agarose acts as a sieve
Typically resolve 200 bp-20 kbp
fragments <200 bp, polyacrylamide gels
fragments> 20 kbp, pulse field gels
Include DNA size standards
Factors That Effect Mobility Of DNA
Fragments In Agarose Gels
Agarose Concentration
Higher concentrations of agarose facilitate separation of
small DNAs, while low agarose concentrations allow
resolution of larger DNAs (Remember-inversely
proportional!)
Voltage
As the voltage applied to a gel is increased, larger
fragments migrate proportionally faster that small
fragments
Charge is evenly spread (uniform) so the larger
fragments will have more charged groups
Factors That Effect Mobility Of DNA
Fragments In Agarose Gels
Electrophoresis Buffer
The most commonly used for double stranded
(duplex) DNA are TAE (Tris-acetate-EDTA) and
TBE (Tris-borate-EDTA).
Effects of Ethidium Bromide
Staining dye that inserts (intercalates) into the DNA
between the nitrogenous bases (“rungs of the ladder”)
and glows when exposed to UV light
Binding of ethidium bromide to DNA alters its mass
and rigidity, and therefore its mobility
Comparison of Agarose Concentrations
% agarose: 2% 4% 5%
500 bp
500 bp
200 bp
200 bp
500 bp
200 bp 50 bp
50 bp
50 bp
Fragment Resolution: Agarose Gel
Electrophoresis
% Agarose DNA fragment,
kb
0.5 30-1
0.7 12-0.8
1.0 10-0.5
1.2 7-0.4
1.5 3-0.2
Gel Electrophoresis: The Basics
The movement of molecules is impeded in the
gel so that molecules will collect or form a band
according to their speed of migration.
The concentration of gel/buffer will affect the
resolution of fragments of different size ranges.
Genomic DNAs usually run as a “smear” due to
the large number of fragments with only small
differences in mass
Agarose Electrophoresis of Restriction
Enzyme Digested Genomic DNA
A B
Gel Electrophoresis: Apparatus and
Types of Gels
Horizontal Gel Units (“Submarine Gels”)
Most DNA and RNA gels
Agarose
Vertical Gel Units
Polyacrylamide gels
Typically sequencing gels
Pulsed Field Gel Units
Any electrophoresis process that uses more than one
alternating electric field
Agarose
Large genomic DNA (Chromosomal)
Electrophoresis Equipment: Horizontal or
Submarine Gel
DNA/RNA is negatively charged: RUN TO RED
Agarose Gel Electrophoresis
DNA/RNA is negatively charged: RUN TO RED
Agarose Gel Electrophoresis
Horizontal Gel Format
Reservoir/Tank
Power Supply
Casting Tray and Combs
www.biorad.com
Agarose Gel Apparatus
Electrophoresis Equipment: Vertical Gel
Vertical Gel Format: Polyacrylamide Gel
Electrophoresis
Reservoir/Tank
Power Supply
Glass Plates, Spacers, and Combs
www.biorad.com
Polyacrylamide Gel Electrophoresis
(PAGE)
Electrophoresis Equipment
Combs are used to put wells in the cast
gel for sample loading.
Regular comb: wells separated by an “ear” of
gel
Houndstooth comb: wells immediately adjacent
PULSE FIELD GEL ELECTROPHORESIS
APPARATUS
Types Of Pulsed Field Gel Electrophoresis
Field inversion gel Transverse alternating field
Crossed field Contour-clamped
(Reverse) homogeneous electric
field
Pulsed Field Gel Electrophoresis
Used to resolve DNA molecules larger than 25
kbp
Periodically change the direction of the electric
field
Several types of pulsed field gel protocols
FIGE: Field inversion gel electrophoresis
TAFE: Transverse alternating field electrophoresis
RGE: Crossed field electrophoresis
CHEF: Contour-clamped homogeneous electric field
Preparation Of Intact DNA For PFGE
Conventional techniques for DNA purification
(organic extraction, ethanol precipitation)
produce shear forces
DNA purified is rarely greater than a few
hundred kb in size
This is clearly unsuitable for PFGE which can
resolve mb DNA
The problem of shear forces was solved by
performing DNA purification from whole cells
entirely within a low melting temperature (LMT)
agarose matrix
Preparation Of Intact DNA For PFGE
Intact cells are mixed with molten low melting
point (LMT) agarose and set in a mold forming
agarose ‘plugs’
Enzymes and detergents diffuse into the plugs
and lyse cells
Proteinase K diffuses into plugs and digests
proteins
If necessary restriction digests are performed in
plugs (extensive washing or PMSF treatment is
required to remove proteinase K activity)
Plugs are loaded directly onto PFGE and run
Using PFGE In The Molecular Investigation Of
An Outbreak Of S. marcescens Infection In An
ICU
An outbreak due to S. marcescens infection was
detected in the ICU
A total of 25 isolates were included in this study:
12 isolates from infected patients
nine isolates from insulin solution
one isolate from sedative solution
one isolate from frusemide solution
two isolates from other wards which were epidemiologically-
unrelated
Singapore Med J 2004 Vol 45(5) : 214
Using PFGE in the Molecular Investigation Of An Outbreak of
S. marcescens Infection in an ICU
Singapore Med J 2004 Vol 45(5) : 214
Polyacrylamide Gel Electrophoresis
(PAGE)
PAGE is the preferred method for
PROTEINS but can be used for DNA/RNA
Gel prepared immediately before use by
copolymerization of acrylamide and N,N'-
methylene bis acrylamide under UV light.
Porosity controlled by proportions of the
two components.
Larger pore size for larger proteins.
Gradient gels also possible.
Electrophoresis of Nucleic Acids
Polyacrylamide Gel Electrophoresis (PAGE)
Advantages
High degree of resolving power.
Can effectively and reproducibly separate
molecules displaying 1 bp differences in
molecular size.
Optimal separation is achieved with nucleic
acids that are 5–500 bp in size.
Electrophoresis of Nucleic Acids
Polyacrylamide Gel Electrophoresis (PAGE)
Typical Conditions
Vertical gel setup, TBE buffer
(Tris-borate/EDTA) and constant power.
Disadvantages
Acrylamide monomer is a neurotoxin.
Polyacrylamide gels can be difficult to handle.
Electrophoresis of Nucleic Acids
Agarose Gel Electrophoresis
Advantages
Greater range of separation of nucleic acid
molecules.
Optimal separation is achieved with nucleic
acids that are 200 bp to 30 kb in size.
Ease of preparation and handling.
PAGE: Critical Parameters
Polymerization reaction critical
High grade acrylamide, bis-acrylamide
Break down into acrylic acid (long shelf life
solutions incorporate inhibitors of
polymerization)
Must have even heat distribution to
prevent “smiling”
Polymerization Of Polyacrylamide
PAGE: DNA
High resolution of low molecular weight
nucleic acids (500bp)
Polymerization of acrylamide monomers
into long chains
Cross link chains with bis-acrylamide
Initiated by free radicals in ammonium
persulfate, stabilized by TEMED
Pore size determined by % acrylamide
Electrophoresis of Nucleic Acids:
Polyacrylamide Gel Electrophoresis
(PAGE)
Typical Conditions
Vertical gel setup, TBE buffer
(Tris-borate/EDTA) and constant power.
Disadvantages
Acrylamide monomer is a neurotoxin.
Polyacrylamide gels can be difficult to handle.
PAGE Fragment Resolution: Denaturing
Conditions (6M Urea)
% Fragment Bromophenol Xylene
Acrylamide Size Blue Cyanol
30 2-8 6 20
20 8-25 8 28
10 25-35 12 55
8 35-45 19 75
6 45-70 26 105
5 70-300 35 130
4 100-500 ~50 ~230
PAGE Fragment Resolution: Non
Denaturing PAGE
% Fragment Bromophenol Xylene
Acrylamide Size Blue Cyanol
3.5 100-1000 100 460
5.0 100-500 65 260
8.0 60-400 45 160
12.0 50-200 20 70
20.0 5-100 12 45
Polyacrylamide Gel Electrophoresis of
Restriction Digested PCR Products
FII SNP
FV SNP
