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Principles of Dna Isolation & Purification
Principles of Dna Isolation & Purification
Principles of Dna Isolation & Purification
Blood
Buccal cells
Cultured cells
Bacterial plasmids
Biopsies
Forensic samples i.e. body fluids, hair
follicles, bone & teeth roots.
Blood
(White blood cells)
Semen
(Sperm cells)
Hair with roots
(Hair follicle cells)
Skin, dandruff
(Skin cells)
Vaginal fluids(Mucosal surfaces)
Nasal secretions (Mucosal surfaces)
Urine
(Mucosal surfaces)
Feces
(Digestive system cells)
Biological Sources:
All nucleated cells.
Extraction
Blood
Semen
Hair Root
Saliva
Bone
Basic rules
Blood first lyse (explode) the red blood cells with a
gentle detergent such as Triton-X-100.
Wash cells haemoglobin (and other pigments) inhibits
restriction enzymes and TAQ polymerase.
Work on ice to slow down enzymatic processes.
Wear gloves to protect your samples from you!!
DNA Isolation
Basic Steps
Cell lysis
Removal of proteins
- protease
- adsorption or extraction
DNA precipitation by ethanol
DNA dilution in water or buffer
DNA Isolation
Four Basic Methods
1. Phenol-chloroform extraction
(different solubility conditions in solvents)
2. Salting out method
(protein precipitation by NaCl)
3. Adsorption method
(silica-gel membrane)
4. Denaturation of proteins by heating
Cells
Extract
BacterialCells
Ortissueculturecells
Orblood
Orflies..
HOW?
Organic
extraction
PureDNA
3 Phenol Extraction
gentle rocking several
hours
4 Ethanol Precipitation
5 RNAse followed by
proteinase K
6 Repeat Phenol Extrac-tion
and EtOH ppt
Detail of step 3
Phenol Extraction
mix sample with equal volume
of sat. phenol soln
retain aqueous phase
optional chloroform/isoamyl
alcohol extraction(s)
aqueous phase
(nucleic acids)
phenol phase
(proteins)
Extraction/Precipitation Method
Step 3: Organic extraction
Mix thoroughly with
an equal volume of
organic solvent
e.g. phenol,
chloroform, or
phenol:chloroform
Aqueous
Centrifuge
3 Phenol Extraction
gentle rocking several
hours
4 Ethanol Precipitation
5 RNAse followed by
proteinase K
6 Repeat Phenol Extrac-tion
and EtOH ppt
Detail of step 4
EtOH Precipitation
2-2.5 volumes EtOH, -20o
high salt, pH 5-5.5
centrifuge or spool out
Extraction/Precipitation Method
Step 4: Nucleic Acid Precipitation
Before
After
After
70% EtOH
Supernatant
Centrifuge
Wash
Centrifuge
Pellet
Dissolve
pellet (H2O,
TE, etc.)
Detail of step 5
Using Nucleases to Remove Unwanted DNA or RNA
Add DNase
+ DNase (protein)
Add RNase
+ RNase (protein)
Quantity from UV
Spectrophotometry
DNA and RNA absorb maximally at 260
nm.
Proteins absorb at 280 nm.
Background scatter absorbs at 320 nm.
Quantity from UV
Spectrophotometry
[DNA] =
(A260 A320) X dilution factor X 50 g/Ml
[RNA] =
(A260 A320) X dilution factor X 40 g/mL
Concentration = g of DNA or RNA per mL
of hydrating solution
Volume of
hydration
solution
DNA yield
DNA purity
The purity of the DNA is reflected in the
OD260 : OD 280 ratio and must be
between 1.6 and 2.00.
Decreased 260:280 ratio means that
contaminating protein is still present.
Repurify sample.
Total RNA:
1% to 2% gel, 0.125 g/ml ethidium bromide in
gel and/or in running buffer
Electrophorese at 80100 volts, 2040 minutes.
Gel Electrophoresis
Separation method
Gel
sieve structure of polymer molecules with pores
Gel
- agarose
- polyacrylamid
Gel Electrophoresis
Separation method
PRINCIPLE:
the movement of charged molecules in electric field
the movement direction from to +
(the nucleic acids consist of
negatively charged phosphate groups)
DNA-rate in gel depends on DNA-fragment length
in indirect proportion
The length of unknown fragments is compared to
the length of standard fragments
RFLP: Electrophoresis
DNA is visualized using electrophoresis
Negatively charged DNA moves through a gel with a
current
Smaller DNA moves faster than larger DNA fragments
Lambda DNA
1 kb ladder
Lambda DNA cut
with Hind III 12,218 bp
23,130 bp
100 bp ladder
6,018 bp
9,416 bp
3,054 bp
1,500 bp
1,000 bp
6,557 bp
2,036 bp
600 bp
4,361 bp
1,636 bp
48,500 bp
(48.5 kb)
300 bp
1,018 bp
100 bp
2,322 bp
2,027 bp
517 bp
Lambda DNA
marker
summary
Sample for DNA extraction
Lysis of cells at elevated temperature +
detergent + enzyme in salt buffer
Removal of cellular proteins
Precipitation of nucleic acids with ethanol
Quantitation and purity measurement of
DNA
POLYMERASE CHAIN
REACTION (PCR)
molecular Xeroxing
The process by which scientists make
millions of copies of specific pieces of
DNA.
The size of these Xeroxed DNA
fragments differ from one person to
another.
Denatured
Heat
DNA
Double Helix
Uncoiled
Heat
Anneal
Primers
?
Heat
2 copies of original template-DNA has doubled!
After 30+
heating/cooling
cycles, we have
oodles of DNA
?
2 cycles
3 cycles
4 cycles
Biological
Material
Capillary
Electrophoresis
Extract/Purify
DNA
Visualize with
Fluorescent
Scanner
(More Obsolete
Technology)
Amplify using
PCR
Result analysis
IF specific band visualize at 1500 bp, then
may identify this Condyloma acuminate tissue
was infected by corresponding type HPV.
Fig.1
Amplification of HPV 11 L1gene by PCR
1. marker:DNA/HindIII and EcoRI
2.3.4. L1 gene PCR products(1.5kb)
5. negative control
Paternity Cases
Whos your daddy?
1.
2.
1.
2.
Homicide or
Rapes:
OJ Simpson
RFLP: Autoradiograph