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Chapter 5: Resolution and Detection of Nucleic Acids
Donna C. Sullivan, PhDDivision of Infectious Diseases
University of Mississippi Medical Center
Objectives
Explain the principle and performance of electrophoresis as it applies to nucleic acids.
Compare and contrast agarose and polyacrylamide gel polymers.
Explain the principle and performance of capillary electrophoresis as it is applies to nucleic acid separation.
Describe the general types of equipment used for electrophoresis.
Discuss methods and applications of pulsed field gel electrophoresis.
Compare and contrast detection systems used in nucleic acid applications.
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 LaboratorySizing 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 ElectrophoresisAgarose 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 sieveTypically 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
500 bp
200 bp
50 bp
% agarose: 2% 4% 5%
500 bp
200 bp
50 bp
500 bp
200 bp
50 bp
Comparison of Agarose Concentrations
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
A B
Agarose Electrophoresis of Restriction Enzyme Digested Genomic DNA
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
Pulse 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
www.biorad.com
Reservoir/TankPower Supply
Casting Tray and Combs
Agarose Gel ElectrophoresisHorizontal Gel Format
Agarose Gel Apparatus
Electrophoresis Equipment: Vertical Gel
www.biorad.com
Reservoir/TankPower Supply
Glass Plates, Spacers, and Combs
Vertical Gel Format: Polyacrylamide Gel Electrophoresis
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 Pulse Field Gel Electrophoresis
Field inversion gel Transverse alternating field
Crossed field (Reverse)
Contour-clamped homogeneous electric
field
Pulse 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
Critical Parameters: Pulse Field Gel ElectrophoresisDepend on time it takes molecules of
various sizes to change directions in a gelSmall DNA molecules are sieved (pass
through the pores in the agarose gel)Large DNA molecules are not “sieved” but
“squeezed” through the gel at about the same rate, called the limiting mobility
Size of Fragments and Distance Traveled Not Linear When Large Fragments Are Analyzed
Movement Of DNA In Gels
Pulse Field Gel Electrophoresis
PFGE works by periodically altering the electric field orientation
The large extended coil DNA fragments are forced to change orientation
Size dependent separation is re-established because the time taken for the DNA to reorient is size dependent
Comparison of Migration: Horizontal vs. CHEF
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+
-
- -
+
++
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
FIGE GEL
CHEF: Contour-Clamped Homogenous Gel Electrophoresis Based on hexagonal array of alternate
electric fields at 120 degree angle Generates a more uniform electric field
when compared to other PFGE systems Programmable, autonomously controlled
electrodes Extremely versatile system based on CHEF hexagonal
array All electrophoretic parameters can be controlled at each
electrode Can generate electric field and switching characteristics
of any PFGE system
PFGE of Bacterial DNA
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
Using PFGE in the molecular investigation of an outbreak of S. marcescens infection in an ICU The S. marcescens from patients, insulin
solution and sedative solution showed an identical PFGE fingerprint pattern.
The isolate from the frusemide solution had a closely-related PFGE pattern to the outbreak strain with one band difference.
Found that the insulin and sedative solutions used by the patients were contaminated with S. marcescens and the source of the outbreak.
Singapore Med J 2004 Vol 45(5) : 214
Comparison Of Agarose Gel And PFGE
Panel B: Agarose gel electrophoresis
Panel C: PFG electrophoresis
Pulsed Field Gel Electrophoresis was applied to the study of Duchenne Muscular Dystrophy. Since the DMD gene is 2.3Mbp, it was necessary to use PFGE in order to uncover the genetic defect. The use of PFGE analysis on patients with the disease soon revealed that in 50% of the cases large deletions or duplications were a responsible for the disease (Mathew, 1991).
Polyacrylamide Gel Electrophoresis (PAGE)PAGE is the preferred method for
PROTEINS but can be used for DNA/RNAGel 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 AcidsAgarose Gel ElectrophoresisAdvantages
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)
% Acrylamide
Fragment Size
Bromophenol Blue
Xylene 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
% Acrylamide
Fragment Size
Bromophenol Blue
Xylene 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
FV SNP
FII SNP
Polyacrylamide Gel Electrophoresis of Restriction Digested PCR Products
Denaturation of DNA: Urea and Formamide
Both urea and formamide effectively lower the melting point of the DNA molecules, allowing the structures to fall apart at lower temperatures.
Preparation of Polyacrylamide Gel
Pour into glass plate gel sandwich and polymerize.
Prepare DNA samples by adding loading buffer.
Document and verify loading order of samples and electrophoretic conditions (voltage).
Stain gel, visualize DNA, photograph/ document and dispose of gel properly.
FV SNP
FII SNP
PAGE of Restriction Digested PCR Products