Resolution and Detection of Nucleic Acids

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

DNA/RNA is negatively charged: RUN TO RED

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

(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.
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.
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.
 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:
(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

Resolution and Detection of Nucleic Acids

Uploaded by

Copyright:

Available Formats

Resolution and Detection of Nucleic Acids

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Resolution and Detection of Nucleic Acids

Uploaded by

Copyright:

Available Formats

Resolution and Detection of

Electrophoresis is the movement of

Nucleic acid moves from a negative to a

DNA and RNA are

 The gel itself is composed of either

 Separates fragments based on mass,

DNA/RNA is negatively charged: RUN TO RED

DNA/RNA is negatively charged: RUN TO RED

Regular comb: wells separated by an “ear” of

Houndstooth comb: wells immediately adjacent

Crossed field Contour-clamped

 Conventional techniques for DNA purification

Singapore Med J 2004 Vol 45(5) : 214

Singapore Med J 2004 Vol 45(5) : 214

 Polymerization reaction critical

 High resolution of low molecular weight

% Fragment Bromophenol Xylene

% Fragment Bromophenol Xylene

You might also like

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.