PRINCIPLES OF DNA

ISOLATION & PURIFICATION

DNA can be isolated from any

nucleated cell.
DNA is a giant anion in solution.

Sources of DNA include

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

Some Important Definitions

Buffer solutions

Chelation

Salting Out

Basic Principle
Sampling
Cell suspension
Cell lysis
Purification
Precipitation

List of chemicals used in DNA extraction

Tris
EDTA
SDS
Proteinase K
NaCl
Phenol
Chloroform
Isoamyl Alcohol
Isopropanol
Ethanol

The Mammalian Blood

Red Blood Cells. Anucleate. about 4 million to 6 million
per cubic millimeter (microliter) of blood.
White Blood Cells. Nucleate. about 4,00011,000 per
cubic millimeter (microliter) of blood.
Platelets. Anucleate. about 150,000400,000per cubic
millimeter (microliter) of blood.

Outline of DNA extraction

There are five basic steps in DNA extraction from blood
Remove red blood cells and membrane proteins.
Remove cellular and histone proteins bound to the DNA.
Precipitate DNA in cold ethanol or isopropanol, DNA is
insoluble in alcohol and clings together.

Out Line Continued.

Wash the resulting DNA pellet with alcohol

Solubilize the DNA in a slightly alkaline buffer

______________________

EDTA

(Ethylenediaminetetraacetic acid)

Chemical formula C10H16N2O8

Chelating agent

Carboxylic acid groups

amine groups

EDTA As Anti-Coagulant

anticoagulant

Tetradenate ligand

Can be problematic

pH 8.0

Tris

trishydroxymethylaminomethane

Molecular formula;
C4 H11 NO3
Biological buffer.
An alkalizer

SDS

Sodium dodecyl sulfate

Chemical formula;
C12H25NaO4S
ionic surfactant

disrupt the phospholipid

bilayer
Disrupts non-covalent bonds
in the proteins

Removal of RBCs and other cell debris

Effect of freezing blood samples

Osmotic dehydration

Proteinase K Hammers away

Proteins
Tritirachium album
The predominant site of cleavage is the peptide bond adjacent to
the carboxyl group of aliphatic and aromatic amino acids.
In general, proteins require denaturation and disulfide bond
cleavage before enzymatic digestion can go to completion.
Proteinase K displays strong proteolytic activity on denatured
proteins and on native proteins as well.

Continued..

Calcium is a stabilizer of Proteinase K.

if the EDTA-Ca2+ complex is removed from the enzyme solution by

gel filtration, a total of 80% of the enzyme activity is lost.

pH range of 4.312.0, with optimal activity at pH 8.0.

Retains >80% of its activity at temperatures of 2060C

Getting rid of the protein

Organic solvent extraction using equal volume TE-sat.
phenol:chloroform:Iso-amyl alcohol (25:24:1)
protein at the interface after centrifugation

followed by extraction with chloroform:iso-amylalcohol to

remove phenol
OR

Salt-out proteins by precipitation with NaCl or Na-acetate

protein pelleted after centrifugation.

Continued

When the salt concentration is increased the protein

molecules coagulate by forming hydrophobic
interactions with each other.
The salt shields the negative phosphate end of DNA
which allows these ends to come closer so they can
precipitate out of a cold alcohol solution .

Ethanol precipitation

Washing with 70% Ethanol

DNA Extraction by Organic Method

phenol
Chloroform.
Isoamyl alcohol

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