Capillary Electrophoresis Flashcards
1
Q
what is an allelic ladder
A
- 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
2
Q
what is an anode buffer container
A
- positive charge
- has a built-in overflow chamber to maintain constant fluid height
3
Q
what is a buffer
A
- 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
4
Q
what are capillaries and what happens inside them
A
- 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
5
Q
what is acapillary array
A
- 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
6
Q
explain capillary electrophoresis
A
- 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
7
Q
what causes carry-over and how do you solve it
A
- 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
8
Q
what is a cathode buffer container
A
- labyrinth design on one side(B) contains water/rinse reagents
- other side (A) contains buffer
- held in the autosampler
9
Q
what are some CE denaturants and what do they do
A
- 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
10
Q
what is a charge-coupled device camera (CCD)
A
- 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
11
Q
what are the components of a CE instruments
A
- capillary filled with polymer
- cathode + anode buffer vials
- two electrodes
- high-voltage power supply
- laser
- fluorescence detector (CCD)
- sample tray
- computer
12
Q
what is a conditioning reagent
A
- used to prime the polymer pump, during water washes, and during an instrument shut down
- one-time use
13
Q
what causes cross-talk and how can it be solved
A
- 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
14
Q
what is the detection cell heater block
A
holds the detection cell in place for laser detection
15
Q
what is the detection cell window
A
- fragile
- allows dye-tagged fragments to be detected
16
Q
explain the detection of fluorescence
A
- 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
