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Transcript
DNA Methodologies
• Sterilization
– Clean the workstation with alcohol and bleach.
– Autoclaving and ultraviolet light (UV radiation).
• Consumables and reagents.
• Equipment
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–
–
–
–
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Pipettes- P20, P200, P1000
Block heater
Vortex
Centrifuge
PCR machine
Electrophoresis box and power supply
DNA sequencing machine
Computer
Tissue Sample
DNA Extraction
DNA Quantitation
PCR mtDNA
PCR STRs
Genetic Analyzer- mtDNA sequencing and STR analysis
Analysis of Results
Conclusion/s
DNA Protocol
• DNA Extraction
–
–
–
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FTA Card
Chelex
Spin columns
Organic- simple and
differential.
• DNA Quantitation
– Direct, non-blot
– Direct, slot-blot
– Real Time PCR
• PCR Amplification
– Polymerase Chain Reaction
• DNA Sequencing or Typing
• Analysis and Interpretation
Human Cell
How do we get the nuclear and mitochondrial DNA
out of the cell?
DNA Extraction Protocols #1
• FTA Card
– Pipette aliquot on
card.
– WBCs lyse on paper
and DNA is trapped.
– Use hole punch to
remove paper for
DNA analysis.
– Wash.
– Analysis.
FTA Protocol
DNA Extraction Protocols #2
• Chelex
– Incubate blood sample
in 5% Chelex at 56°C
for 30 min.
– Boil for 8 min.
– Centrifuge to remove
inhibitors and cellular
debris.
DNA Extraction Protocols #3
• Column Extraction
– Lyse cells and digest proteins.
• Physical, heat, detergent
• Proteinase K
– Bind DNA to silica membrane by
centrifugation.
– Wash with 70% ethanol by
centrifugation.
– Elute DNA with TAE (Tris-Acetate
- EDTA) or water by
centrifugation.
DNA Extraction Protocols #4
• Organic Extraction
– Lyse cells and digest proteins.
• Physical, heat, detergent
• Proteinase K
– Organic extraction.
• Phenol-chloroform
– Centrifuge to remove supernatant.
– Precipitate DNA.
• Ethanol or isopropanol
– Centrifugation to pellet DNA.
– Elute DNA with TAE or water.
Differential
Extraction
• Technique used to separate
sperm cells from non-sperm
cells (epithelial cells).
– Vaginal swabs
1. Epithelial cells are lysed with
a mild extraction buffer
containing Sodium Dodecyl
Sulfate (SDS).
2. Centrifugation to pellet sperm
cells, DNA from epithelial
cells is in the supernatant.
3. Lyse sperm cells using
Dithiothreitol (DTT).
DNA Quantitation
• DNA Quantitation
– Direct, non-blot
– Direct, slot-blot
– Real Time PCR
• Why quantitate?
– U.S. FBI Standards require it!
– 1ng-2.5ng yields consistent typing results.
• 1 cell= 6.1pg (164 cells needed for analysis)
– Too much DNA leads to artifacts and too much signal.
– Too little DNA leads to allelic dropout.
DNA Quantitation #1
• Direct, non-blot
– AluQuant (Promega Corp.)
• Human-specific probe that binds to
Alu insertions (highly repetitious
DNA).
• Luciferin-luciferase reaction and a
luminometer to detect the amount of
light.
• Emission is compared against
standards.
– Yield Gel
• Standards of known concentration are
compared against a sample.
DNA Quantitation #2
• Direct, slot-blot
– QuantiBlot (Applied
Biosystems)
• Human-specific probe
(D17Z1) binds to DNA.
• Chemiluminescence is
used to determine the
DNA concentration
against a set of standards.
DNA Quantitation #3
• Real Time PCR
– Quantifiler (Applied Biosystems)
• Human genes are amplified.
• Gene number doubles after each cycle.
• Each cycle yields more fluorescence.
• Fluorescence is recorded and compared against standards to
determine DNA concentration.
PCR Product Amount is Proportional to the Amount
of Input DNA Template
Exponential PCR
During the exponential expansion of the
PCR the amount of product produced is
proportional to the amount of template. Here
we show the total amount of product
following 32 cycles.
1.00E+10
9.00E+09
ng product
8.00E+09
7.00E+09
6.00E+09
2ng template
1ng template
0.5ng template
5.00E+09
4.00E+09
3.00E+09
2.00E+09
1.00E+09
0.00E+00
0
5
10
15
20
# Cycles
25
30
35
Develop a standard curve
5.0 ng
1.3 ng
0.31 ng
0.078 ng
Ct
0.0 ng
(reagent blank)