Transcript Chapter 5: Resolution and Detection of Nucleic Acids

Chapter 5: Resolution and
Detection of Nucleic Acids
Donna C. Sullivan, PhD
Division 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
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
50 bp
200 bp
50 bp
50 bp
Fragment Resolution: Agarose Gel
Electrophoresis
% Agarose
0.5
0.7
1.0
1.2
1.5
DNA fragment,
kb
30-1
12-0.8
10-0.5
7-0.4
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
 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
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 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
Electrophoresis
Depend on time it takes molecules of
various sizes to change directions in a gel
Small 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 reestablished because the time taken for the
DNA to reorient is size dependent
Comparison of Migration: Horizontal vs.
CHEF
-
-
+
-
-
+
+
+
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 epidemiologicallyunrelated
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/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 (Trisborate/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
Acrylamide
Size
Blue
30
20
10
8
6
5
4
2-8
8-25
25-35
35-45
45-70
70-300
100-500
6
8
12
19
26
35
~50
Xylene
Cyanol
20
28
55
75
105
130
~230
PAGE Fragment Resolution: Non
Denaturing PAGE
%
Fragment Bromophenol Xylene
Acrylamide
Size
Blue
Cyanol
3.5
5.0
8.0
12.0
20.0
100-1000
100-500
60-400
50-200
5-100
100
65
45
20
12
460
260
160
70
45
Polyacrylamide Gel Electrophoresis of
Restriction Digested PCR Products
FII SNP
FV SNP
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.
PAGE of Restriction Digested PCR
Products
FII SNP
FV SNP