an overview of capillary electrophoresis electrophoresis the movement of (charged)...

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An Overview of Capillary Electrophoresis

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Page 1: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

An Overview of Capillary Electrophoresis

Page 2: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

ElectrophoresisElectrophoresis

The movement of (charged) The movement of (charged) particles/fragments through a mediumparticles/fragments through a medium..Cations migrate toward the negatively charged electrode (cathode) and anions are attracted toward the positively charged electrode (anode).

Affected by:Affected by:

•SizeSize

•Shape/Conformation of FragmentShape/Conformation of Fragment

•pHpH

•Matrix/MediumMatrix/Medium

•TemperatureTemperature

Page 3: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 4: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 5: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 6: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 7: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 8: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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 homopolymer8M urea5% 2-pyrrolidinon 100 mM N-Tris-(hydroxymethyl)methyl-3-aminopropane-sulfonic acid (TAPS) at pH 8.0

Page 9: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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

Page 10: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

Inlet (cathode)

Outlet (anode)

Capillary Electrophoresis (CE)

Argon Ion Laser

Fill with Polymer Solution

Fill with Polymer Solution

50 m x 47 cm50 m x 47 cm

15 kV15 kV

- +Burn capillary window

Data AcquisitionData Acquisition

DNA Separation occurs in

~30 minutes...

DNA Separation occurs in

~30 minutes...

Page 11: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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

Page 12: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

Multiple Instrumentation Options for Different Sample Throughput

310 1 cap

3100-Avant

4 cap

3100 16 cap

Page 13: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

ABI PRISM® 310 Genetic Analyzer

Autosampler

Capillary

Heat Plate

Syringe Pump

DetectionWindow

Page 14: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

Electrokinetic Injection

Page 15: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 16: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

Sample Tube

DNA-

-

Electrokinetic Injection Process

Electrode

CapillaryD

NA

-

-

The amount of salt in the sampleaffects how much DNA migratesinto the capillary.

Amount of DNA is inversely Proportional to the ionic strength.

Page 17: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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

Page 18: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 19: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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

Page 20: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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

Page 21: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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

Page 22: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

CCD Chip Color DetectionCCD Chip Color Detection

“VIRTUAL FILTERS”

SPATIALAXIS

SPECTRAL AXIS

Page 23: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

Detection

• 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

Page 24: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

Detection Methods

Fluorescence

Label one of the primers for each locus

Page 25: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 26: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 27: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

excitation emission

Stokes Shift

Page 28: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively
Page 29: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

520 540 560 580 600 620 640WAVELENGTH (nm)

100

80

60

40

20

0

5-FAM JOE NED ROX

Laser excitation(488, 514.5 nm)Laser excitation(488, 514.5 nm)

Normalized Fluorescent Intensity

Fluorescent Emission Spectra for ABI Dyes

ABI 310 Filter Set F

Page 30: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

Dye Set G5

Emission Spectra of 5-dye Set

0

20

40

60

80

100

500 550 600 650 700

Wavelength (nm)

No

rmalized

em

issio

n

6FAM™ dye VIC™ dye NED™ dye

PET™ dye LIZ™ dye

Page 31: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 32: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

4- and 5-dye Matrices Generated on the Same ABI PRISM® 310 Genetic Analyzer

4-dye Matrix Table

5-dye Matrix Table

Page 33: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

Troubleshooting Capillary Electrophoresis

• Offscale data• Migration problems• Laser problems• Buffer depletion• Capillary failure• Low or no current• Spikes in baseline

Page 34: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

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.

Offscale data

Page 35: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

Pull-up

Due to signal and spectral overlap

Pull-up

Page 36: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 37: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 38: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

Migration Problems

Page 39: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 40: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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

Page 41: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 42: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 43: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 44: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 45: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively
Page 46: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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)

Page 47: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively
Page 48: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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.

Page 49: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively
Page 50: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

Troubleshooting

The operator manuals for the Capillary Electrophoresis instruments have an extensive section on troubleshooting bad data due to problems with the instrument.

Page 51: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

ABI PRISM® 3100 Genetic Analyzer

Page 52: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

ABI PRISM® 3100-Avant Genetic Analyzer: Doors Open

Page 53: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

Instrument Open

•Forced air oven with two internal fans to provide thermal control and uniformity

•Thermostat inside oven for continuous monitoring

•Internal instrument lighting

Page 54: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

4-Capillary Array

Page 55: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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

Page 56: An Overview of Capillary Electrophoresis Electrophoresis The movement of (charged) particles/fragments through a medium. Cations migrate toward the negatively

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