Electrophoresis

Introduction to Electrophoresis

.

What is Electrophoresis? A method of separating large molecules (such as DNA fragments or Proteins ) from a mixture of similar molecules. An electric current is passed through a medium containing the mixture, and each kind of molecule travels through the medium at a different rate, depending on its electrical charge and size. Separation is based on these differences. Agarose and acrylamide gels are the media commonly used for electrophoresis of proteins and nucleic acids

Gel Electrophoresis

• The pH and other buffer conditions are arranged so that the molecules being separated carry a net (negative) charge so that they will me moved by the electric field toward the positive pole. As they move through the gel, the larger molecules will be held up as they try to pass through the pores of the gel, while the smaller molecules will be impeded less and move faster. This results in a separation by size, with the larger molecules nearer the well and the smaller molecules farther away.

Electrophoresis of DNA• The Phosphate groups on the backbone of

the DNA molecule readily give up their H+

ions, therefore nucleic acids are negatively charged in most buffer systems.

• DNA molecules will migrate away from the negative electrode (cathode), and migrate towards the positive electrode (anode).

• The higher the voltage, the greater the force felt by the DNA molecule, and the faster they will migrate in an electric field.

Electrophoretic Separation of DNA

• Agarose Gel Electrophoresis

• Acrylamide Gel Electrophoresis (Native versus Denaturing Conditions)

• Capillary Electrophoresis

Gel Matrices Used for Electrophoresis of DNA

• Agarose Gels have fairly large pore sizes and are used for separating larger DNA molecules (Restriction Fragment Length Polymorphism Analysis)

• Polyacrylamide Gels are used to obtain high resolution separations for smaller DNA molecules (STR analysis and DNA sequence analysis)

Introduction to Agarose Gel Electrophoresis

Agarose Gel Electrophoresis

• Yield Gel – Semiquantitative and qualitative analysis of isolated DNA

• Separation of DNA restricted with Hae III (RFLP analysis) followed by a Southern Blot and Hybridization with a labeled probe

• Post Amplification confirmation and qualitative assessment of PCR product

Assessing DNA Quality

Experiment:

100 ng K562 DNA

Digest with DNAse

Molecular Weight Ladder

~23Kbp

~ 2kbp

Time

0153045

seconds

123

minutes


• An electrophoresis chamber and power supply

• Gel casting trays, which are available in a variety of sizes and composed of UV-transparent plastic.

• Sample combs, around which molten agarose is poured to form sample wells in the gel.

• Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).

Agarose Gel Electrophoresis• Loading buffer, which contains

something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow monitoring or how far electrophoresis has proceeded.

• A fluorescent dye used for staining nucleic acids, such as Ethidium bromide, Sybr Green, or Sybr Gold.

• Transilluminator or Fluorescent Gel Scanner for photodocumentation

Migration of DNA Fragments in Agarose

• Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of

their molecular weight

Agarose Concentration

• By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose facilite separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs.

Agarose Concentration

Agarose (%)

Range of separation of linear DNA (in kilobases)

0.3 60 - 5

0.6 20 - 1

0.7 10 - 0.8

0.9 7 - 0.5

1.2 6 - 0.4

1.5 4 - 0.2

2.0 3 - 0.1

Agarose ElectrophoresisVoltage

• As the voltage applied to a gel is increased, larger fragments migrate proportionally faster that small fragments. For that reason, the best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel (the cm value is the distance between the two electrodes, not the length of the gel).

Electrophoresis Buffer• Several different buffers have been recommended

for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity. If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel! Similarly, if you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it.

Agarose Gel Electrophoresis System

Agarose Gel Tips• When preparing agarose for electrophoresis, it is

best to sprinkle the agarose into room-temperature buffer, swirl, and let sit at least 1 min before microwaving. This allows the agarose to hydrate first, which minimizes foaming during heating.

• Electrophoresis buffer can affect the resolution of DNA. TAE (Tris-Acetate-EDTA) buffer provides better resolution of fragments >4 kb, while TBE (Tris-Borate-EDTA) buffer provides better resolution of 0.1- to 3-kb fragments. In addition, use TBE buffer when electrophoresing >150 V and use TAE buffer with supercoiled DNA for best results.

Agarose Gel Tips

• Migration of DNA is retarded and band distortion can occur when too much buffer covers the gel. The slower migration results from a reduced voltage gradient across the gel..

• Loading DNA in the smallest volume possible will result in sharper bands.

• Electrophoresing a gel too "hot" can cause the DNA to denature in the gel. It can also cause the agarose gel to deform.

Weigh out ~ a gram of agarose.

• Mix the agarose with 50- 100 ml of buffer.

• Heat to dissolve the agarose.

• Assemble the gel tray and comb.

Pour the gel.

• Load one DNA sample into each well on the gel.

Connect the gel to a low voltage power supply.


• After the samples are loaded, slowly fill the gel box with the 1X buffer. Make sure the gel is completely covered.

• Alternatively gels can be covered with buffer first, and then samples in dye buffer are loaded into each well.


• Turn the switch on the power supply to "Off" before connecting the electrophoresis chamber.

• Place the lid tightly on the chamber and plug the electrical leads into the recessed output jacks of the power supply.

• Plug the red (+) lead into the red jack, and the black (-) lead into the black jack.


• Select the desired voltage on the power supply. A voltage of 150 will permit the electrophoresis run to be completed in about an hour.


• Turn the power supply switch "ON." The blue migration dye should move toward the positive electrode (red). If it is migrating toward the negative electrode (black), turn off the power supply, remove the lid, turn the gel tray 180o, replace the lid, and turn the power supply "ON."

After completion of the run, add a DNA staining material and visualize the DNA

under UV light.

Ethidium Bromide

Ethidium Bromide

• This compound contains a planar group that intercalates between the stacked bases of DNA.

• The orientation and proximity of ethidium with the stacked bases causes the dye to display an increased flourescence compared to free dye (in solution).

• U.V. radiation at 254 nm is absorbed by the DNA and transmitted to the bound dye.

• The energy is re-emitted at 590 nm in the red-orange region of the spectrum.

Ethidium Bromide

• Ethidium bromide is usually prepared as a stock solution of 10 mg/ml in water, stored at room temp and protected from light.

• The dye is usually incorporated into the gel and running buffer, or conversely, the gel is stained after running by soaking in a solution of ethidium bromide (0.5 ug/ml for 30 min).

• The stain is visualized by irradiating with a UV light source (i.e. using a transiluminator) and photographing with Polaroid film.

• The usual sensitivity of detection is better than 0.1 ug of DNA.

Gel Staining

Introduction to Polyacrylamide Gel Electrophoresis

Polyacrylamide Gel Electrophoresis

Monomeric acrylamide (which is neurotoxic) is polymerized in the presence of free radicals to form polyacrylamide. The free radicals are provided by ammonium persulphate and stabilized by TEMED (N'N'N'N'-tetramethylethylene-diamine). The chains of polyacrylamide are cross-linked by the addition of methylenebisacrylamide ( bis) to form a gel whose porosity is determined by the length of chains and the degree of crosslinking.


• The chain length is proportional to the acrylamide concentration : usually between 3.5 and 20%. Cross-linking BIS-acrylamide is usually added at a ratio of 2g BIS : 38g acrylamide (1 : 20).

Polyacrylamide gels are poured between two glass plates held apart by spacers of 0.4 - 1.0 mm and sealed with tape. Most of the acrylamide solution is shielded from oxygen so that inhibition of polymerization is confined to the very top portion of the gel. The length of the gel can vary between 10 cm and 1m depending on the separation required. They are always run vertically with 0.5x or 1x TBE as a buffer.


• Polyacrylamide gels have enough resolving power to separate fragments differing by only one base pair in size, but their range is ~ 5 to 1000 bp. They are much more difficult to handle than agarose gels.


Types of Polyacrylamide gels

• Non-denaturing gels : these are run at low voltages - 8V/cm - and 1 x TBE to prevent denaturation of small fragments of DNA by the heat generated in the gel during electrophoresis. The rate of migration is approximately inversely proportional to log10 of their size. However, the base sequence composition can alter the electrophoretic mobility of DNAs such that two DNAs of the same size may show up to a 10% difference in electrophoretic mobility

• Denaturing gels : these gels are polymerized with a denaturant that suppresses base pairing in nucleic acids - this is usually urea but can be formamide. Denatured DNA migrates through the gel at a rate which is almost completely independent of its composition or sequence.

Types of Polyacrylamide gels

% acrylamide (w/v) Effective range of

with BIS at 1:20 separation - bp

3.5 1 000 - 2 000

5.0 80 - 500

8.0 60 - 400

12.0 40 - 200

15.0 25 - 150

20.0 6 - 100

Acrylamide Gel Electrophoresis

Effect of Gel Percentage and Size Separation

% Polyacrylamide gel

Bromophenol blue

Xylene cyanol (41)

5 35 130

6 26 106

8 19 76

10 12 55

12 8 28

Dye Migration in Different % Polyacrylamide Gels

SEPARATION OF PCR PRODUCTS

DENATURING ACRYLAMIDEGEL ELECTROPHORESIS

Digital Imaging HardwareFMBIO® II Fluorescence Imaging System

PowerPlexTM 1.1

P-41411 P-41414

CSF1PO

TPOX

TH01

vWA

D16S539

D7820

D13S317

D5S818

P-41411 P-41414M C AF M C AF M C AF M C AF

Introduction to Capillary Electrophoresis

Electrophoresis• Electrophoresis refers to the migration of

charged electrical species when dissolved, or suspended, in an electrolyte through which an electric current is passed. Cations migrate toward the negatively charged electrode (cathode) and anions are attracted toward the positively charged electrode (anode). Neutral solutes are not attracted to either electrode. The traditional electrophoresis equipment offered a low level of automation and long analysis times.

• Detection of the separated bands was performed by post-separation visualization.

• The analysis times were long as only relatively low voltages could be applied before excessive heat formation caused loss of separation.

Electrophoresis

Heat Dissipation

• In conventional slab gel electrophoresis the Joule heat associated with the generation of current during separation can cause problems of peak dispersion. This Joule heat causes the formation of convention currents within the gel which mixes the zones during separation and results in band broadening and peak dispersion. Heat generation therefore restricts the operating voltages that can be used in slab gel electrophoresis which produces longer analysis times.

Heat Dissipation

• Performing electrophoresis in a capillary allows the heat to be effectively dissipated through the capillary walls which reduces any convection related band broadening. This improved heat dissipation means that higher operating voltages can be used in CE which can produce significantly faster analysis times.

Capillary Electrophoresis

• The advantages of conducting electrophoresis in capillaries was highlighted in the early 1980's by the work of Jorgenson and Lukacs who popularized the use of CE. Performing electrophoretic separations in capillaries was shown to offer the possibility of automated analytical equipment, fast analysis times and on-line detection of the separated peaks.


• Heat generated inside the capillary was effectively dissipated through the walls of the capillary which allowed high voltages to be used to achieve rapid separations. The capillary was inserted through the optical center of a detector which allowed real time capillary detection.


• Operation of a CE system involves application of a high voltage (typically 10-30kV) across a narrow bore (25-100m) capillary. The capillary is filled with electrolyte solution which conducts current through the inside of the capillary. The ends of the capillary are dipped into reservoirs filled with the electrolyte.


• Electrodes made of an inert material such as platinum are also inserted into the electrolyte reservoirs to complete the electrical circuit. A small volume of sample is injected into one end of the capillary. The capillary passes through a detector, usually a UV absorbance detector, at the opposite end of the capillary.


• Application of a voltage causes movement of sample ions towards their appropriate electrode usually passing through the detector. The plot of detector response with time is generated which is termed an electropherogram.

(prior to separation of fluorescent dye colors)

Raw Data from the ABI Prism 310

Capillaries

• The capillaries used are normally fused silica capillaries covered with an external polyimide protective coating to give them increased mechanical strength as bare fused silica is extremely fragile. A small portion of this coating is removed to form a window for detection purposes. The window is aligned in the optical centre of the detector.

Capillaries

• Capillaries are typically 25-75 cm long with 50 and 75 micron being the most commonly employed inner diameters. On standard commercial CE instruments the capillary is often held in a housing device such as a cartridge to facilitate ease of capillary insertion into the instrument and to protect the delicate detection window area.

Capillaries

• The inner surface of the capillary can be chemically modified by covalently binding (coating) different substances onto the capillary wall. These coatings are used for a variety of purposes such as to reduce sample adsorption or to change the ionic charge on the capillary wall.

Capillary Gel Electrophoresis:

The capillaries we typically use in CE are commercially available in single or multiple arrays. We use capillaries that range about 30 to 50 centimeters in length, 0.150 to 0.375 millimeters in outer diameter, and a 0.010 to 0.075 millimeter diameter channel down the center.

Capillary Electrophoresis Process


ABI Prism 310 Genetic Analyzer

ABI Prism 310 Genetic Analyzer

capillary

Syringe with polymer solution

Autosampler tray

Outlet buffer

Injection electrode

Inlet buffer

Chemistry Involved• Injection

– electrokinetic injection process– importance of sample preparation (formamide)

• Separation– capillary– POP-4 polymer– buffer

• Detection– fluorescent dyes with excitation and emission traits – virtual filters (hardware/software issues)

Sample Detection

CCD Panel

ColorSeparation

Ar+ LASER (488 nm)

Fluorescence ABI Prism spectrograph

Capillary or Gel Lane

Size Separation

Labeled DNA fragments (PCR products)

Detection region

Principles of Sample

Separation and Detection

Sample Application

• The standard sample injection procedure is to dip the capillary and electrode into the sample solution vial and to apply a voltage. If the sample is ionized and the appropriate voltage polarity is used then sample ions will migrate into the capillary. This type of injection is known as electrokinetic sampling.

SAMPLE APPLICATION

Sample Tube

DNA-

-

Electrokinetic Injection Process

Electrode

CapillaryD

NA

-

-

Q is the amount of sample injected

r is the radius of the capillary

cs is the sample concentration

E is the electric field applied

t is the injection time

s is the sample conductivity

b is the buffer conductivity

ep is the mobility of the sample molecules

eo is the electroosmotic mobilityRose et al (1988) Anal. Chem. 60: 642-648

Q =s

r2cs(ep + eo)Etb

Comments on Sample Preparation

• Use high quality formamide (<100 S/cm)!– ABI sells Hi-Di formamide– regular formamide can be made more pure with ion

exchange resin

• Deionized water vs. formamide– Biega and Duceman (1999) J. Forensic Sci. 44: 1029-1031

– water works fine but samples are not stable as long as with formamide

• Denaturation with heating and snap cooling– use thermocycler for heating and wet ice bath to snap

cool– heat/cool denaturation step is not always necessary...

Separation Issues

• Run temperature -- 60 oC helps reduce secondary structure on DNA and improves precision

• Electrophoresis buffer -- urea in running buffer helps keep DNA strands denatured

• Capillary wall coating -- dynamic coating with polymer

• Polymer solution -- POP-4, POP-6

DNA Separation Mechanism

+-DNA-

DNA-

DNA-DNA- DNA-

• Size based separation due to interaction of DNA molecules with entangled polymer strands

• Polymers are not cross-linked (as in slab gels)• “Gel” is not attached to the capillary wall• Pumpable -- can be replaced after each run• Polymer length and concentration determine the separation

characteristics

Detection Issues

• Fluorescent dyes– spectral emission overlap– relative levels on primers used to label PCR

products– dye “blobs” (free dye)

• Virtual filters– hardware (CCD camera)– software (color matrix)

Filters determine which wavelengths of light are collected onto the CCD camera

Filters determine which wavelengths of light are collected onto the CCD camera

Laser Used in ABI 310

• Argon Ion Laser• 488 nm and 514.5 nm for excitation of dyes• 10 mW power• Lifetime ~5,000 hours (1 year of full-time use)• Cost to replace ~$5,500• Leads to highest degree of variability between

instruments and is most replaced part• Color separation matrix is specific to laser used on

the instrument

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Electrophoresis

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