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