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An Overview of Capillary
Electrophoresis
Electrophoresis
The movement of (charged)
particles/fragments through a medium.
Cations migrate toward the negatively charged electrode (cathode) and anions
are attracted toward the positively charged electrode (anode).
Affected by:
•Size
•Shape/Conformation of Fragment
•pH
•Matrix/Medium
•Temperature
Capillary Electrophoresis
• Operation of a CE system involves application of a
high voltage (typically 10-30kV) across a narrow bore
(25-100µm) capillary.
• The capillary is filled with a viscous polymer solution
which serves as the sieving medium.
• The ends of the capillary are dipped into reservoirs
filled with the electrolyte.
Capillary Electrophoresis
• 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.
Capillary Electrophoresis
• 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.
Capillaries
• The capillaries used are normally fused silica
capillaries covered with an external polyimide
protective coating to give them increased mechanical
strength
– 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 center of the detector.
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 forensic capillaries typically used in CE from ABI are
47cm x 50um - Uncoated. 36cm from the cathode to the
detector
With Performance Optimized Polymer 4% (POP4)
composed of:
4%DMA homopolymer
8M urea
5% 2-pyrrolidinon
100 mM N-Tris-(hydroxymethyl)methyl-3aminopropane-sulfonic acid (TAPS) at pH 8.0
DNA Separation Mechanism
-
DNA - DNADNA
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
+
Capillary Electrophoresis (CE)
Fill with Polymer
Solution
Argon Ion
Laser
50 m x 47 cm
-
Burn capillary
window
+
DNA Separation occurs in
Inlet
(cathode)
15 kV
~30 minutes...
Data Acquisition
Outlet
(anode)
How does CE work?
• Samples amplified, denatured
• Electrokinetic injection – gets sample into capillary
• Electrophoresis - size separation of DNA
• Detection – Excitation of dyes and collection of
emission wavelengths
• Software analysis
Multiple Instrumentation Options
for Different Sample Throughput
310
1 cap
3100-Avant
4 cap
3100
16 cap
ABI PRISM® 310 Genetic Analyzer
Syringe Pump
Heat Plate
Capillary
Autosampler
Detection
Window
Electrokinetic Injection
Electrokinetic Injection
• The capillary and electrode are placed into the
sample solution vial and a voltage is applied. 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.
Electrokinetic Injection
Process
Capillary
Electrode
The amount of salt in the sample
affects how much DNA migrates
into the capillary.
Amount of DNA is inversely
Proportional to the ionic strength.
-
DNA-
Sample
Tube
Separation
• 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 acts as a sieving medium
to perform the size based separation of DNA
Separation
• DNA molecules posses a constant mass to charge ratio
• Therefore regardless of the size of the DNA molecule
they will have the same force pulling on it when an
electrical field is applied
• A sieving medium can then be used that will retard the
bigger molecules and allow the smaller molecules to
move faster. This allows for a size based separation.
Fluorescent Labeling of PCR
Products
• Dyes are attached to one primer in a pair used to amplify a
STR marker
• Dyes are coupled to oligonucleotides (primers) through
NHS-esters and amine linkages on the 5’end of the primer
usually through a 6-carbon spacer --- Dye-(CH2)6-primer
• Dye-labeled oligonucleotide is incorporated into PCR
product during multiplex PCR amplification giving a specific
color “tag” to each PCR product
• Dyes can be spectrally distinguished using virtual filters
and CCD imaging to yield different colored peaks in ABI 310
electropherogram
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
Labeled DNA fragments
(PCR products)
Principles of Sample Separation
and Detection
Capillary or
Gel Lane
Sample Detection
Size
Separation
Ar+
LASER
CCD Panel
(488 nm)
Color
Separation
Detection
region
Fluorescence
ABI Prism
spectrograph
CCD Chip Color Detection
“VIRTUAL FILTERS”
SPATIAL
AXIS
SPECTRAL AXIS
Detection
Filters determine which wavelengths of light are
collected onto the CCD camera
• Virtual filters
– hardware (CCD camera)
– software (color matrix)
Detection Methods
Fluorescence
Label one of the primers for each locus
Fluorescence
Fluorescence results when a fluorescent dye
(fluorophore) absorbs incident light
(excitation) and in response emits light (emission) at
a different wavelength
– During excitation a photon from a laser source excites
fluorophore electron to an excited state.
– The electron then undergoes a conformational change and the
excited electron emits a photon at a lower energy as it returns to
the ground state.
Fluorescence
• Because energy and wavelength are inversely
proportional to each other the emission photon has a
higher wavelength than the excitation photon
• The difference in wavelength between the fluorescence
excitation maximum and fluorescence emission
maximum is called the Stokes shift.
• The Stokes shift allows the use of optical filters to
separate excitation light from emission light.
Stokes Shift
excitation
emission
Fluorescent Emission Spectra for ABI Dyes
5-FAM JOE NED
ROX
ABI 310 Filter Set F
100
80
60
40
20
0
520 540 560
580 600 620
Laser excitation
WAVELENGTH (nm)
(488, 514.5 nm)
640
Dye Set G5
Normalized emission
Emission Spectra of 5-dye Set
100
80
60
40
20
0
500
550
600
650
700
Wavelength (nm )
6FAM™ dye
VIC™ dye
PET™ dye
LIZ™ dye
NED™ dye
Fluorescence Measurements
• Spectral overlap occurs because there are regions
between different dyes that share the same wavelength.
• Multicomponent analysis is performed with a
mathematical matrix that subtracts out the contributions of
other dyes in each measured fluorescent dye.
• One method to perform multicomponent analysis is to
examine a standard set of DNA fragments labeled with
each dye. This is known as matrix standard samples.
• The computer software analyzes the data from each of
the dyes and creates a matrix file to reflect the color
overlap between the various dyes.
4- and 5-dye Matrices Generated on the
Same ABI PRISM® 310 Genetic Analyzer
4-dye Matrix Table
5-dye Matrix Table
Troubleshooting Capillary
Electrophoresis
•
•
•
•
•
•
•
Offscale data
Migration problems
Laser problems
Buffer depletion
Capillary failure
Low or no current
Spikes in baseline
Offscale data
If too much sample DNA is added to the PCR reaction mixtures, the
fluorescence intensity from the PCR products may exceed the linear dynamic
range for detection by the instrument. This is referred to as “off-scale” data.
Multicomponent analysis cannot be performed accurately on data that is
off-scale. Samples with off-scale peaks will exhibit raised baselines and/or
excessive “pull-up” of one or more colors under the off-scale peaks.
Analyzed data from off-scale peaks should not be used for quantitative
comparisons. For example, the stutter peak that corresponds to an off-scale
main peak is likely to be overestimated.
Pull-up
Pull-up
Due to signal and spectral overlap
Migration Problems
• Migration problems can be visualized when samples on
the same run in different injections migrate at a different
rate from each other.
• This is best seen when the Ladder migrates differently
from samples. The ladder is used to assign allele calls to
the samples.
• If the samples and ladder are not sized within the same
range then the samples peaks will not have the correct
allele calls.
• One cause of migration problems is fluctuations in
temperature during a run.
Migration Problems
• The instruments are equipped with a hot plate or oven to
keep the temperature constant during the
electrophoresis process.
• However there are regions of the capillary that are not in
contact with the heat source. If there are variations in the
room temperature during the course of a run, samples
will migrate at different rates.
• High temperatures will cause samples to migrate faster
and colder temperatures will cause samples to migrate
slower.
Migration Problems
Laser Problems
• The laser used in the genetic analyzers is an Argon ion
laser.
• Overtime a condition known as outgassing can occur.
Outgassing is the slow release of gas (argon) from the
laser tube.
• This results in a loss of laser power.
• As the laser power reduces so does the resulting signal
produced by the DNA molecules. The result is low peak
heights which will continue to get lower over time.
Buffer depletion
• If there is a drop off in the current this can be caused by
buffer depletion.
• During electrophoresis ions move through the capillary.
• Positive ions move to the negatively charged electrode
and negative ions to the positively charged electrode.
• This ion movement results in an imbalance referred to as
buffer depletion
• To avoid this from happening the buffer should be
changed regularly
Capillary Failure
• Abnormally broad peaks due to loss of resolution can be
caused by capillary failure.
• Capillary failure occurs as a result of DNA and enzymes
from the injected samples adhering to the capillary wall.
• To prevent this Applied Biosystems does not recommend
using a capillary past 100 injections.
• Many laboratories have validated capillaries for use well
past 100 injections.
Capillary Failure
• A good way to check to see if the problem is due to
capillary failure or a bad sample will be to look at the size
standard peaks.
• If the size standard peaks are as they should be but the
samples peaks are low or appear to have lost resolution
then their may be a problem with the way the sample
was set up.
• Also if both the size standard and sample peaks are bad
in one injection but there are no problems in other
injections that one sample may have been injected
badly.
Low or No current
• There are several problems which can cause low or no
current.
• A common problem is if the capillary ends are not stored
in water or buffer when the instrument is not in use.
• If the ends of the capillary dry out then urea or other
salts from the buffer will form crystals at the capillary
ends.
• These crystals can partially or fully clog the capillary
ends resulting in low or no current. This will be seen as
consistent low level data.
Low or no current
• Low or no current can also be caused by bubbles in the
pump block.
• These air bubbles block the flow of current from one
electrode to the next.
• The pump block should always be checked to ensure no
bubbles are present in pump block or in base of syringe
since an air bubble there may be injected into the pump
block.
Dye Blobs
• Free dye (not coupled to primer) can be injected into the
CE capillary and interfere with detection of true STR
alleles
• Dye blobs are wider and usually of less intensity than
true STR alleles (amount depends on the purity of the
primers used)
• Dye blobs usually appear at an apparent size that is
unique for each dye (e.g., HEX ~170 bp)
Spikes in Baseline
• Spikes in baseline appear as peaks but they usually
have a sharp point and are in all dye colors.
• They can be caused by fluctuations in the current,
precipitates in the polymer or old polymer.
Troubleshooting
The operator manuals for the Capillary Electrophoresis
instruments have an extensive section on
troubleshooting bad data due to problems with the
instrument.
ABI PRISM® 3100 Genetic Analyzer
ABI PRISM® 3100-Avant Genetic
Analyzer: Doors Open
Instrument Open
•Forced air oven with two
internal fans to provide
thermal control and uniformity
•Thermostat inside oven for
continuous monitoring
•Internal instrument lighting
4-Capillary Array
3100- Avant System Overview
• 4 capillary instrument with upgrade option to
16-capillaries without taking additional bench
space
• Based on 3100 technology
• 16-capillary instrument = 3100 system
3100 System Overview
• Dual side illumination
• Temperature range 18-65º C
• Autosampler supports 96 and 384 trays (two trays per
set up)
• Up to 24 hours unattended operation