Capillary Electrophoresis Flashcards
what is an allelic ladder
- amplified product provided with the CE kit and run with each plate
- contains the most common alleles for all loci and is combined with the ILS
- assigns allele calls, determines genotypes, and accounts for shifts in migration
what is an anode buffer container
- positive charge
- has a built-in overflow chamber to maintain constant fluid height
what is a buffer
- supplies charge ions to conduct the electrophoretic current across the capillary
- dissolves the polymer and stabilizes the DNA
- if not refilled, the current can fluctuate
- buffer depletion impairs separation of DNA fragments
what are capillaries and what happens inside them
- must be kept in deionized water or buffer or they will dry out and will become clogged, affecting results
- inside capillary
- DNA moves in an electrophoretic flow from the cathode (-) to the anode (+)
- flexible polymer chains are navigated by the DNA
- positive buffer ions line the negative capillary walls
what is acapillary array
- capillaries are long, narrow glass tubes with a polyimide coating
- part of the capillary has the polyimide coating burned off to act as the detection window
- a cathode surrounds each capillary
- polymer is pumped into the capillaries before every injection
- area where DNA separation occurs
explain capillary electrophoresis
- prior to sample injection, the capillary array is filled with polymer solution
- electrokinetic injection uses a low voltage to inject the samples into the instrument
- separation occurs using platinum electrodes (cathodes), a polymer (sieve), buffers, and denaturants
- detection occurs when the fluorescent dyes are excited by a laser and documented by the CCD camera
what causes carry-over and how do you solve it
- occurs when DNA from the previous injection transfer into the sample is injected with the same capillary in the next injection
- in groups of three; well A1 capillary goes in well A4
- usually caused by an over-concentrated sample
- can look like a low level profile/contaminant on the egram
- can be solved by re-injecting
what is a cathode buffer container
- labyrinth design on one side(B) contains water/rinse reagents
- other side (A) contains buffer
- held in the autosampler
what are some CE denaturants and what do they do
- amplicons (PCR products)
- diluted in a mixture of formamide and internal size standard
- formamide
- helps reduce salt levels and denature double-stranded DNA
- LSPCL uses high quality formamide (Hi-Di) with low conductivity
- breaks down when exposed to air
- internal size standards
- used for DNA sizing to aid in allele designation
what is a charge-coupled device camera (CCD)
- a photosensitive device that measures light intensity emitted from fluorophores and converts it to an electronic signal (RFUs)
- filters collect the light on the camera
- different wavelengths are detected at different locations
- a photon striking the detector is converted to electric signal, and the intensity is reported in RFUs
- more intensity = more DNA = high peaks = larger RFU scale
- located behind the detect window
what are the components of a CE instruments
- capillary filled with polymer
- cathode + anode buffer vials
- two electrodes
- high-voltage power supply
- laser
- fluorescence detector (CCD)
- sample tray
- computer
what is a conditioning reagent
- used to prime the polymer pump, during water washes, and during an instrument shut down
- one-time use
what causes cross-talk and how can it be solved
- occurs when fluorescent signal from one sample interferes and is detected in the adjacent sample/capillary
- usually caused by an over-concentrated sample
- can look like irregular morphology or like a low level profile
- can be solves by re-plating
what is the detection cell heater block
holds the detection cell in place for laser detection
what is the detection cell window
- fragile
- allows dye-tagged fragments to be detected
explain the detection of fluorescence
- detection is performed automatically by measuring the time span from sample injection to sample detection
- uses fluorescent dyes with excitation and emission traits
- a fluorescent dye is attached to the 5’ end of the primer and incorporated into the target region of the DNA
- a laser is directed through the detection window, exciting the dyes as they pass the window
- each fluorescent dye emits fluorescence at a different wavelength, corresponding to different colors (400-600nm)
- a CCD camera separates the fluorescence of each dye by color
explain DNA separation
- electrokinetic injections produce narrow injection zones that allow DNA separations to occur in a short distance
- smaller fragments arrive at the detection point first
- speed of migration correlates to base pair size (via the internal size standard)
- the color of the dye with the base pair size indicate the locus
- base pair size and the locus indicate allele designation
explain electrokinetic injection
- an electric voltage is applied while the end of the capillary is immersed into the DNA sample
- current is generated by the voltage
- negative DNA moves towards the positive anode at the end of the capillary because it is repelled by the negative cathode in the sample well
- the negative cathode surrounds each capillary
- improper sample prep can introduce negative salts into the DNA, impacting electrokinetic injection
what is electrophoresis
- movement of charged molecules through a medium into an electric field
- detects DNA based on dye and its corresponding marker to determine the allele
- negatively charged DNA moves from the negative cathode to the positive anode
- separates DNA by speed through the capillary and by color using 51 dye-labeled fragments per contributor
- conducted on single-stranded DNA
what causes erratic current in CE
can be caused by lack of anode buffer, bubbles, slow leak, poor quality reagents, and arcing(?)
what causes fluorescent contamination
caused by poor quality reagents, infrequent cleaning, or contamination
what are some/all of the genetic analyze components
- buffer
- capillary array
- CCD camera
- conditioning reagent
- detection cell heater block
- detection cell window
- high voltage power supply
- laser
- oven & oven door
- oven condensation reservoir
- polymer delivery pump
- polymer pouch
what is the purpose of high voltage power supply
provides voltage that is applied after the sample is injected into the capillary
what are some parts of the genetic analyzer that can have an impact on DNA separation/temperature
- capillary
- polymer
- buffer
- temperature
- voltage
what laser do the 3500XL analyzers use
solid state diode laser excitation source (505 nm)
what causes loss of resolution
can be caused by air bubbles, poor capillaries, poor quality reagents, leak in capillary, impurities, poor instrument maintenance, too much sample
what causes low signal
can be caused by air bubbles, lack of current, low sample volume, no size standard, clogged capillaries, bad laser, poor quality reagents, incorrect sample prep, and contamination
what causes migration
can be caused by old polymer, poor reagents, old buffer, incorrect buffer levels, changes in room temperature, bubbles, polymer leaks, and contamination
what is the purpose of the oven & oven door
maintains uniforms capillary array temperature
what is the purpose of the oven condensation resevoir
collects condensation from the oven
what is the polymer that the genetic analyzers use
- POP-4: Performance Optimized Polymer 4%
- contains a high concentration of urea to help keep DNA denatured
- provides a sieve for DNA to move through the capillary
what does the polymer delivery pump (PDP) do
pumps polymer into the array and allows for automated maintenance procedures
what is the polymer pouch
- lasts through five plates/20 injections
- contains additional volume for bubble remover and priming abilities
what are the three requirements for STR typing
- dna sizing precision
- spatial resolution
- spectral resolution
what is the purpose of the size standard (WEN)
- aka internal lane standard (ILS)
- contains DNA fragments of known base pair size to correlate with results from run to run
- injected with each sample as part of master mix
- utilized to determine size of unknown sample fragments
explain sizing precision of DNA
- must be consistent so that samples can be related to allelic ladders and yield reproducible results
- requires a size standard and an allelic ladder
what is spatial resolution and why is it necessary
- prior to running instrument
- determines the relationship between the signal emitted from each capillary and where it is detected by the CCD camera
- necessary to separate DNA fragments by a single nucleotide
- maps the position of each capillary to a region on the camera
what is spectral resolution and why is it necessary
- prior to running instrument
- generates a matrix for each capillary for the software to separate out any overlap
- separates dye colors so that PCR products of the same color can be related to different loci
- spectral calibration is unique to each instrument
- if calibration doesn’t work properly, uneven baseline or pull-up can occur
what causes spikes
can be caused by dust, dried polymer, dried buffer, poor quality formamide, air bubbles, electrical surges, and poor capillaries
why is temperature important in a genetic analyzer
- stable temperature is required for the genetic analyzer
- variations in temperature affect DNA migration through the capillaries
explain the two types of electrophoresis
- gel electrophoresis
- old method
- capillary electrophoresis
- full automated, can be left unattended
- multiple samples can be run at once
- quicker separation and no cross contamination
why is voltage important in a genetic analyzer
consistent voltage is needed during and between every run
