Kingsley Odigie and Calvin Lee
Team 4
Exam II Summary
CE Talk---Butler & McCord
 23 pairs of chromosomes
 3 billion base pairs---1/(1/4)3,000,000,000 possible combinations
 DNA analysis by RFLP—long process, requires high quality and quantity of
DNA
 Kary Mullis invented PCR
 STR and other genetic variabilities: Give reproducible results from day to day;
resolution of a single base over the range of analysis; precision under 0.17 base
pair for size separation; stability over time; insensitivity to matrix effects;
relative accuracy though not absolute
 Methods of determination of variability: probe hybridization; charge based
mobility & separation—gel & capillary electrophoresis; partition & ion
exchange—HPLC; Conformation—SSCP, heteroduplex polymorphism; size
measurement—mass spectrometry
 Variation in the methods: Probe based methods—can be difficult to detect
length variation; HPLC—lacks resolution; MS—has trouble with sizes above 90
bp; Conformational polymorphisms—will not always vary sufficiently;
Electrophoresis—currently best option, but trouble with precision & resolution
 Issue: although PCR is rapid & efficient, sample loads are increasing---untested
rape kits nationwide is between 180,000 to 500,000
 PCR—Application—has improved speed & detection capabilities; automated
system needed to increase sample throughput
Capillary Electrophoresis
 Benefits of capillary electrophoresis: injection, separation and detection are
automated; rapid separations are possible and peak information is automatically
stored for easy retrieval
 Electrophoresis process: injection; separation and detection
 Charges of DNA vary with size
 Electroosmotic flow & electrophoretic flow responsible for separation
 The electric field strength influences the shape of the DNA
 To improve resolution: lower field strength; increase polymer concentration;
increase polymer length and use long capillary
 Know separation issues; capillary wall coating; electrophoresis buffer; polymer
solution and run temperature
 Injection methods for CE---Hydrodynamic and Electrokinetic
 Detection Issues
• Fluorescent dyes
– spectral emission overlap
– relative levels on primers used to label PCR
products
– dye “blobs” (free dye)
• Virtual filters (determine which pixels are
used
– hardware (CCD camera)
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Kingsley Odigie and Calvin Lee
Team 4
Exam II Summary
–
software (color matrix)
 Laser Used in ABI 310
• Argon Ion Laser
• 488 nm and 514.5 nm for excitation of dyes
• 10 mWpower
• 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
NOTE
• There are no filters in a 310
• Its just the choice of pixels in the CCD detector
• All the light from the grating is collected
• You just turn some pixels on and some off
 What affects precision?
Lots of things:
– Temperature
– Sequence of Rox standard vs sample
– Sequence of allele vs ladder
– Conformation of DNA
– Polymer matrix
– Capillary condition
– Buffer concentration
– pH
The Fluorescence Process
 Three-stage process: Stage 1 : Excitation—caused by photon of energy supplied
by an external source such as an incandescent lamp or a laser; Stage 2 : ExcitedState Lifetime--The excited state exists for a finite time (typically 1–10 × 10-9
seconds); Stage 3 : Fluorescence Emission--A photon of energy is emitted as the
fluorophore is returned to its ground state S0. Due to energy dissipation during the
excited state.
 Fluorescence Detection---Four essential elements of fluorescence detection:-(1)
an excitation source, (2) a fluorophore, (3) wavelength filters to isolate emission
photons from excitation photons and (4) a detector that registers emission photons
and produces a recordable output, usually as an electrical signal or a photographic
image.
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Kingsley Odigie and Calvin Lee
Team 4
Exam II Summary
 Three types of fluorescence instrument: • A spectrofluorometer measures the
average properties of bulk (μL to mL) samples.; • A fluorescence microscope
resolves fluorescence as a function of spatial coordinates in two or three
dimensions.; • A flow cytometer measures fluorescence per cell in a flowing
stream, allowing subpopulations within a large sample to be identified and
quantitated.
Butler Chapter 12
I. Separation Methods
a. Slab Gel
i. How it works
1. Different sized DNA fragments must travel through gel
matrix.
2. Smaller fragments get through faster and larger fragments
take longer.
3. The DNA is propelled by electrical charge; moving to the
positive end. (Remember that DNA is negatively charged.)
4. The gel must be scanned to get quantifiable results.
ii. Types
1. Agarose Gels
a. Seaweed based
2. Polyacrylamide Gels
a. Chemical based gel that allows more control in pore
size. (The smaller the pore size the more the
discrimination between two similar sized strands.)
iii. Advantages
1. Cheap / Low Overhead
2. Prepared gels are available
3. Ability to run multiple gels at the same time
4. Not as temperature sensitive
5. Can be stopped and restarted
iv. Disadvantages
1. Labor intensive / Hand loading of gels
2. Acrylamide gels may contain toxins
3. Requires scanning, an extra process that quantifies the
results accurately
b. Capillary Electrophoresis (CE)
i. How it works
1. Different sized DNA fragments must travel through glass
tube capillary that is filled with viscous polymer solution
that acts like a gel.
2. Smaller fragments get through faster and larger fragments
take longer.
3. The DNA is propelled by electrical charge; moving to the
positive end.
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Kingsley Odigie and Calvin Lee
Team 4
Exam II Summary
4. Detection is done by florescence detection and is directly
translated to a electropherogram, eliminating the need for
scanning.
ii. Advantages
1. Fast (higher voltage = faster run times)
2. Automated (Reduces potential for human error.)
3. Smaller samples are used
4. Reduce possibility of cross-contamination
iii. Disadvantages
1. Expensive
2. Running multiple samples in parallel is less feasible.
3. Temperature sensitive
Butler Chapter 13
I. Fluorescence Detection
a. How it works
i. Fluorescent dye is attached to primer used in PCR
ii. Four different dyes are used
1. Blue, Green, Yellow, Red
2. FAM, JOE, TAMRA, ROX
iii. As the varied length fragments pass through CE tube, toward the
end (finish line) a laser excites dye and its fluorescence is detected.
iv. They will have spectral bleed which means that a red dye (ROX)
will also trigger the Yellow and even the green detector. Most bleed
through will occur in the adjacent color in the spectrum.
v. These readings will be charted in a electropherogram. Silver staning
can also be done for a “light brite”-like gel.
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