Detection and Analysis of Nucleic Acid (Gel Electrophoresis and Gel Documentation) Flashcards
Father of Electrophoresis; did pioneer work on moving boundary electrophoresis (1930) and later developed a zone method for the purification of biomolecules.
Arne Tiselius
- Routinely used for detection and size analysis of proteins and nucleic acids.
- Uses an electrical current
Electrophoresis
_______ : Vertical Gel Electrophoresis (Polyacrylamide Gel)
Proteins
Proteins : _________
Vertical Gel Electrophoresis (Polyacrylamide Gel)
____________ : Horizontal Gel Electrophoresis (Agarose Gel)
DNA and RNA
DNA and RNA : __________________
Horizontal Gel Electrophoresis (Agarose Gel)
DNA and RNA : ____-charged because of phosphor-sugar backbone
Negatively
Migration rate of a molecule depends on two factors:
SHAPE and charge-to-mass ratio
The penetration of the DNA molecules through pores of different sizes provides a _________
Mechanism for separation by molecular mass
DNA electrophoretic mobility is determined by the interplay of three factors
- The relative size of the DNA molecules with respect to the pore size of the
matrix - The effect of the electric field on the matrix
- Specific interactions of the matrix with the DNA molecules during
electrophoresis
3 Parts of Electrophoresis
Voltage, Supporting Medium, and Buffer System
A part of electrophoresis that is responsible for the power supply
Voltage
A part of electrophoresis that consists of a matrix in which the biomolecule separation takes place
Supporting Medium
A part of electrophoresis that conduct electricity by running buffer
Buffer system
Purified linear galactan hydrocolloid isolated from agar or agar-bearing marine algae
Agarose
Alternating copolymer of D-galactose and 3,6-anhydro-L-galactose
Agarose
Agarose is dissolved by ____________
Heating the fibrous powder in an aqueous
solution
Occurs when the fiber bundles become linked together by the formation of additional hydrogen bonds
Gelation
Charge of the Gel Matrix
Negatively-charged
_________ groups in an agarose gel are affixed to the matrix and cannot move
Anionic
Negatively charged residues are surrounded by ____________
Positively charged counterions from the buffer.
The net result is a flow of the solvent toward the cathode, called the ________
Electroosmotic flow
DNA is negatively charged. It migrates in the _____
Opposite direction, toward the anode.
Held together by covalent cross-linking of
Acrylamide and N,N’-methylene bisacrylamide
Polyacrylamide gel structure
Tougher than agarose gels
Polyacrylamide Gel (PAG)
Tougher than agarose gels
Polyacrylamide Gel (PAG)
Used for separating smaller nucleic
acids and proteins
Polyacrylamide Gel (PAG)
2 Layers of Polyacrylamide Gel (PAG) and their pH value
Stacking gel: Top most layer (6.8 pH)
Resolving gel: Lower layer (8.8 pH)
• For denaturing the proteins
• An anionic detergent that breaks
disulfide bonds
Sodium Dodecyl Sulfate-PAGE
• Gives a negative charge to each protein in proportion to its mass • Composed of a hydrophilic group with a net negative charge and a long hydrophobic chain with neutral charge
SDS-PAGE
Factors that Determine the Rate of Migration of a DNA Molecule Through a Gel:
Size of the Molecule Agarose Concentration DNA Conformation Voltage Applied Electrophoresis Buffer Presence of DNA Stains in the Gel Electrophoresis Buffer
Molecular size of nucleic acid is expressed in ___________
Molecular weight equivalent to the number of
bases/ base pairs in the molecule
A linear DNA fragment of a given size migrates at __________ through gels containing different concentrations of agarose
Different Rates
Supercoiled circular DNA, relaxed circular DNA and linear DNA of the same molecular
weight will migrate at _____ through the gel (DNA Conformation)
Different Rates
Rate of migration is ______ to the voltage applied
Proportional
Composition and ionic strength affects DNA mobility; High ionic strength (10X Buffer) –> ______
Electrical Conductance is Efficient
Dyes used to stain DNA in gels are usually intercalating agents; Addition to the gel may _________
retard the rate of migration of the DNA
Materials Required for Agarose Gel Electrophoresis
Electrophoresis Chamber Gel Casting Tray Loading Dye Comb Buffer Agarose
The gel is placed in an ____ which is then connected to a power source. When the electric current is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster.
Electropheresis Chamber
Available in variety of sizes and composed of
UV transparent plastic
The open ends are closed with
tape while the gel is being cast, then
removed prior to electrophoresis
Gel Casting Trays
Placed in the liquid agarose after it has been
poured
Removing it from the hardened gel produces a series of wells used to load the DNA
Comb
Placed in the liquid agarose after it has been
poured
Removing it from the hardened gel produces a series of wells used to load the DNA
Comb
A solution that contains the right ions to
conduct electricity
Running Buffer
Examples of Buffer (4):
TAE (Tris Acetate EDTA)
TBE (Tris Borate EDTA)
Tris phosphate
Tris Citrate Buffer
A mixture of glycerol and tracking dyes
Loading Dye
weighs down DNA sample
by increasing its density
Glycerol
allows visual gauge of
the progress of electrophoresis.
Tracking dye (bromphenol blue; xylene cyanol)
consists of known DNA sizes
used to determine the size of an
unknown DNA sample
DNA ladder
usually contains regularly spaced sized samples which when run on an agarose gel looks like a “ladder”
DNA ladder
Enables estimate DNA size (bp) in the
sample
DNA ladder
Method for Electrophoresis (first 4 parts)
Add 1% of Agarose powder to 25 mL
of 10x TBE
Heat the mixture in a microwave
Add 0.75 uL of loading dye to
the mixture
Pour into casting tray with comb
and allow to solidify
Method for Electrophoresis (second 5 parts)
Remove the Comb
Add Running Buffer and transfer the
agarose gel to the Casting Chamber
Dispense the DNA ladder and the
samples to the loading wells
Run gel at constant voltage until
band separation occurs
View DNA on UV light box and show
results
Detection of DNA: ______________ to view the fluorescent DNA in the gel
UV transilluminator
Quantification of DNA: _______ to DNA being measured
Dye and size must be the same
Evaluation of the Quality of DNA: Bond appear as compact, no _________
double bond or faint band
Characteristics of 1x TAE
contains 40 mM Tris-acetate, and 1 mM EDTA at pH 8.3
provide good resolution of DNA fragments (improved separation of larger size)
recommended for resolution of RNA and DNA fragments larger than 1500 bp, for genomic DNA and for large supercoiled DNA
contain a weak acid, acetic or boric acid
Characteristics of 1x TBE
contains 890 mM Tris base, 890 mM boric acid, 20 mM EDTA at pH 8.3
provide good resolution of DNA fragments (improved separation of smaller size)
contain a weak acid, acetic or boric acid
EDTA is __________, but is added as a preventative because it chelates Mg2+ ions
not absolutely essential
Usually performed at constant voltage
Gel Buffer
The current (measured in mA) increases and _______________
warming of the running buffer occurs.
The voltage is kept low at _______
10V/cm
small- or medium-sized horizontal gel (10–15 cm length)
power supplies are set to between 100 V and 150 V, and gels are run for 50–90 min.
used to monitor the progress of the electrophoresis run
Tracking dyes, also known as loading dyes; or DNA Size Tracking Dye and Density Agents
Ficoll, sucrose, or glycerol, can
increase the density of the sample
The dyes migrate at specific speeds in a given gel concentration and
usually run ahead of the smallest fragments of DNA
Tracking dyes that are commonly used
bromophenol blue and xylene cyanol
Gel loading dye is typically made at _____
6 concentration (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol).
Gel loading dye is typically made at _____
6 concentration (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol).
- routinely used to stain DNA in agarose and polyacrylamide gels
- binds to DNA molecules by intercalating between adjacent base pairs
- powerful mutagen
- limited sensitivity
Ethidium bromide (EtBr)
- used either as a poststain after electrophoresis
* included in the buffer solution
nonmutagenic dyes that stain DNA green, red, or blue are now used in
many laboratories
Some of these dyes require UV irradiation or illumination
The most sensitive dyes are able to detect bands that contain
less than 1ng DNA.
Pulsed electric fields must be applied to the gel to
separate very large DNA molecules
pulsed fields may be applied _______
intermittently in one direction, in opposite directions, or at obtuse angles
The rate at which relaxation occurs depends on ______
DNA size
are trapped in the gel matrix and scarcely move
large DNA molecules
Optimal separation window is determined by ________
the ratio of pulse length to DNA reorientation time
Basic Principle of Pulsed Field
- DNA molecules in pulse field agarose gels are highly dependent on the electric field applied to the gel.
- PFGE can separate DNA molecules up to 10 Mb.
Types of PFGE
Contour-clamped homogeneous electric field (CHEF)
Field-inversion gel electrophoresis (FIGE):
Orthogonal-field alternation gel electrophoresis (OFAGE)
Rotating gel electrophoresis (RGE)
Programmable autonomously controlled electrodes (PACE)
- 4 passive electrodes are arranged hexagonally
- can regulate the voltage on all hexagonally arranged electrodes in a unit electric field.
- the size, location, coordination, stability, and continuity of the electric field are precisely controlled
- DNA molecules as large as 2 Mb can be well separated with a CHEF system alternating between two orientations 120 degrees apart.
- DNA smaller than 50 kb can be separated without distortion.
Contour-clamped homogeneous electric field (CHEF)
- Two fields are arranged in separated straight angle (180 degree)
- can overcome the problem caused by the comigration of nucleic acids and protein-detergent complexes
- provides good resolution, over 800kb
Field-inversion gel electrophoresis (FIGE)
- vertical electrophoresis system
* DNA molecules between 1000 kb and 2000 kb can be separated
Orthogonal-field alternation gel electrophoresis (OFAGE)
- the agarose gel is rotated between two angles periodically
- power supply is turned off after switching the angle during electrophoresis
- easy to change the angle of rotation
- this method is convenient and suitable for the separation of DNA with 50kb to 6000kb.
Rotating gel electrophoresis (RGE)
- 24 electrodes are arranged in a closed contour
- controls all the parameters of the electric field
- flexible, and it is preferable to the other alternating electrophoresis methods
- DNA fragments from 100 bp to more than 6 Mb are separable.
Programmable autonomously controlled electrodes (PACE)
• for molecular typing
• Identification of pathogens in the prevalence of certain diseases (gold
standard method to identification of some bacteria)
• used in epidemiological studies
• used for viral DNA fingerprinting of viruses isolated from the
environment.
Application of PFGE
- carried out in buffers containing entangled hydrophilic polymers that act as the sieving medium.
- offers significant advantages over slab gel DNA separations
CAPILLARY ELECTROPHORESIS
Disadvantage of Capillary Electrophoresis
fragility of the capillaries and capillary coatings and the necessity of running one sample at a time in each capillary
Setup of the Capillary Electrophoresis
• carried out in fused-silica capillaries with a high surface area to volume ratio
• sample is introduced into a buffer-filled capillary either electrokinetically (with
low voltage) or hydrodynamically (with pressure or suction).
• Both ends of the capillary and electrodes are then placed into a buffer solution
that also contains the electrodes, and a high voltage is applied to the system
• The applied voltage causes the analytes to migrate through the capillary and pass
a detector window
Two electrically driven phenomena contribute to the mobility of the analyte:
- electrophoretic mobility of the analyte itself
* electroosmotic flow of the bulk solution.
The net movement of buffer toward the cathode is concurrent with
analyte mobility
Separation Principle: Charge-to-mass ratio
Analytes: Small Ions, small molecules, peptides and proteins, limited DNA
Capillary Zone Electrophoresis
Separation Principle: Isoelectric Point
Analytes: Peptides and Proteins
Capillary Isoelectric Focusing
Separation Principle: Mobility with Buffer
Analytes: Small molecules, Peptides and Proteins
Capillary Isotachophoresis
Separation Principle:
- Charged species - charge-to-mass ratio and micelles, according to hydrophobicity
- Neutral Species - detergent micelle organization based on hydrophobicity
Analytes: Small molecules, peptides, DNA
Micellular Electrokinetic Capillary Chromatography
Separation Principle: Charge-to-mass ratio sieving
Analytes: Peptides and Proteins, DNA
Capillary Gel Electrophoresis Nondenaturing
Separation Principle: Mass Sieving
Analytes: Peptides & Proteins, DNA
Capillary Gel Electrophoresis: Denaturing (SDS, Urea)
- Also known as Gel Doc. System, Gel Image, or Gel Imager
- For Imaging and documentation of nucleic acid
- It is composed of ultraviolet (UV) light transilluminator, a hood and CCTV camera for image capturing
Gel Documentation
Principle of Gel Documentation
- Principle of fluorescence with fluorescent staining of nucleic acids, a fluorescent substance that has bound to nucleic acids is excited by ultraviolet irradiation and emits
fluorescent light.
*The fluorescent substance Ethidium Bromide binds specifically to nucleic acid and the amount of bonding depends on the molecular weight and concentration of the nucleic acid.
Purpose of Gel Documentation
- Photography of stained gel
- Print out of photographic data
- Image data is displayed in real time
- Image still displayed can be printed out with a video printer or saved to a compact flash media for future use.
Different parts in Gel-Doc System
Camera, lenses, filters, overhead illuminator, visible light, and adjustable stage
Camera in Gel Documentation
Ultraviolet camera ranging from 1.4m
upto 8.3m
Computer controlled and motor driven in Gel Documentation
Lenses
Filters:
There is an extensive range of emission
filters use for an array of application
Over head illuminator:
White light, Ultraviolet light, and LED lighting option
For extending the transmitted light application
Visible Light
For extending the transmitted light application
Visible Light
Enables sample to be moved closer to camera
Adjustable Stage
Process for Imaging
Remove - Remove the gel from the plastic gel tray (it slides out).
Place - Place the hood back over the glass panel.
FLIP - flip the switch to turn on the UV light.
TAKE A PHOTO - Click the “snap” button to capture the image.
Process of Cleaning the UV Light Source
- Make sure the UV light is turned off.
- Remove the camera hood.
- Wipe the glass panel with ethanol and a KimWipe.
Important Warnings
- UV light is extremely dangerous. NEVER turn on the light unless the hood covers the ENTIRE glass panel.
- ALWAYS wear gloves when working with gels… ethidium bromide is a carcinogen!
- ALWAYS wear CLEAN gloves when using the keyboard and mouse.
- NEVER dispose of gels in the trash or sink. Use the gel waste bucket
