dna methodologies sterilization –clean the workstation with alcohol and bleach. –autoclaving and...

DNA Methodologies• Sterilization

– Clean the workstation with alcohol and bleach.– Autoclaving and ultraviolet light (UV radiation).

• Consumables and reagents.

• Equipment– 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– 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

0.00E+00

1.00E+09

2.00E+09

3.00E+09

4.00E+09

5.00E+09

6.00E+09

7.00E+09

8.00E+09

9.00E+09

1.00E+10

0 5 10 15 20 25 30 35

# Cycles

ng

pro

du

ct

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.

2ng template

1ng template0.5ng template

0.0 ng

5.0 ng1.3 ng

0.31 ng0.078 ng

Ct

Develop a standard curve

(reagent blank)