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PRINCIPLES OF DNA

ISOLATION & PURIFICATION

DNA can be isolated from any


nucleated cell.

Sources of DNA include

Blood
Buccal cells
Cultured cells
Bacterial plasmids
Biopsies
Forensic samples i.e. body fluids, hair
follicles, bone & teeth roots.

Potential Sources of DNA

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!!

Autoclave all solutions and store in fridge (except SDS


and organic solvents!)
Keep all pellets & supernatants until you have the DNA
you want.

DNA Isolation
Basic Steps

Cell lysis
Removal of proteins
- protease
- adsorption or extraction
DNA precipitation by ethanol
DNA dilution in water or buffer

Getting to the DNA


Cells lyse all cells in presence of :
NaCl so that DNA is stabilised and remains as a double helix,
EDTA which chelates Mg++ and is a co-factor of DNAse which
chews up DNA rapidly,

anionic detergent SDS which disrupts the lipid layers, helps to


dissolve membranes & binds positive charges of chromosomal
proteins (histones) to release the DNA into the solution.

Include a protease (proteinase K) to digest the proteins

incubate the solution at an elevated temperature (56oC to


inhibit degradation by DNAses) for 4-24 hrs.

Getting rid of the protein


Organic solvent extraction using equal volume TE-sat.
phenol:chloroform (24:1)
protein at the interface after centrifugation (10K, 10o, 10)

followed by extraction with chloroform:iso-amylalcohol to


remove phenol
OR
Salt-out proteins by precipitation with NaCl or Naacetate
protein pelleted after centrifugation.

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

Precipitating the DNA


add 2.5 - 3 volumes ice-cold 95%
ethanol/acetate to the DNA & leave at -20oC
overnight.
Centrifuge sample at 10K, 10, 40C.
Wash DNA pellet to remove excess salt in 70%
EtOH and air-dry.
Resuspend in sterile distilled water (sdw) or TE,
pH7.4.
Store at 4oC or frozen at -20oC long term.

Cells
Extract
BacterialCells
Ortissueculturecells
Orblood
Orflies..

HOW?
Organic
extraction

PureDNA

High MW Genomic DNA Isolation


Typical Procedure
1 Harvest cells
2 Cell Lysis
0.5% SDS + proteinase K
(55o several hours)

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

Crude lysate containing


nucleic acids and other
cell constituents

Aqueous

Centrifuge

Collect aqueous phase


Interphase
Organic

Perform additional extractions for increased purity

The aqueous phase contains water-soluble


molecules, including nucleic acids. Proteins and
lipids become trapped in the organic phase,
and are thus separated away. Insoluble debris
become trapped in the interphase between the
two layers

High MW Genomic DNA Isolation


Typical Procedure
1 Harvest cells
2 Cell Lysis
0.5% SDS + proteinase K
(55o several hours)

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

Add alcohol and salt to


precipitate nucleic acids
from the aqueous fraction

Pellet down nucleic acids.


Wash pellet with 70% ethanol to remove
residual salts and other contaminants.
Discard ethanol and allow pellet to dry.

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)

Depending on when nuclease treatment is performed, it may be necessary to


repeat purification steps for protein removal (e.g. phenol/chloroform extraction).

Nucleic Acid Analysis


DNA or RNA is characterized using
several different methods for assessing
quantity, quality, and molecular size.
UV spectrophotometry
Agarose gel electrophoresis
Fluorometry
Colorimetric blotting

DNA Purity and Concentration


Spectrophotometry
Absorbance maximum
for nucleic acids 260 nm
for proteins 280 nm
Concentration of DNA at 260 nm
Purity of DNA: ratio of 260/280 nm

Quantity from UV
Spectrophotometry
DNA and RNA absorb maximally at 260
nm.
Proteins absorb at 280 nm.
Background scatter absorbs at 320 nm.

Quantifying the DNA


The "absorbance" (O.D.) of a chemical is the:
product of its (concentration) x (optical path length) x
(extinction coefficient, E).
Nucleic acids have a peak absorbance in the ultraviolet
range at about 260 nm
1 A260 O.D. unit for dsDNA = 50 g/ml
1 A260 O.D. unit for ssDNA = 33 or 50 g/ml
1 A260 O.D. unit for RNA = 40 g/ml

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

Quantity from UV Spectrophotometry


Calculating Yield
Multiply the concentration of the
DNA or RNA sample by the
volume of hydrating solution added.
Example for DNA: 150 g/mL X 0.1 mL = 15 g
Concentration from UV
Spec. (g DNA 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.

Quality from UV Spectrophotometry

A260/A280 = measure of purity


(A260 A320)/(A280 A320)
1.7 2.0 = good DNA or RNA
<1.7 = too much protein or
other contaminant (?)

DNA Purity and Concentration


Gel Electrophoresis
DNA is stained by intercalating dyes in gel
(Fluorescent dye - ethidium bromide)
Gel is loading with DNA standard
(its concentration is pre-evaluated)
Comparison of two light intensities
standard and our sample

Quality from Agarose Gel


Electrophoresis
Genomic DNA:
0.6% to 1% gel, 0.125 g/mL ethidium bromide
in gel and/or in running buffer
Electrophorese at 7080 volts, 4590 minutes.

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

Ethidium bromide is an intercalating dye,


binds to DNA
It creates complex exciting photons
after UV-exposure

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

DNA Size from Agarose Gel Electrophoresis:


Compares unknown DNA to known size
standards

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

DNA Quality from


Agarose Gel Electrophoresis
High molecular weight band (>48.5 kb)
Smearing indicates DNA degradation (or
too much DNA loaded).

DNA Quality from Agarose Gel


Electrophoresis
Human Whole Blood DNA

Lambda DNA
marker

Whole blood genomic DNA


Lambda DNA cut with
Hind III 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

POLYMERASE CHAIN REACTION


(PCR)
Denature
Template DNA

Heat

Anneal
Primers

Nobel Prize Step


Polymerase
Extension

?
Heat
2 copies of original template-DNA has doubled!

POLYMERASE CHAIN REACTION


(PCR)
1 cycle

After 30+
heating/cooling
cycles, we have
oodles of DNA
?

2 cycles

3 cycles

4 cycles

STEPS: DNA PROFILES FROM


BIOLOGICAL SOURCES

Biological
Material
Capillary
Electrophoresis

Extract/Purify
DNA
Visualize with
Fluorescent
Scanner
(More Obsolete
Technology)

Slot blots vs.


RT-PCR

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