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 4,000–11,000 per
cubic millimeter (microliter) of blood.
• Platelets. Anucleate. about 150,000–400,000per 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 RBC’s 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 4.3–12.0, with optimal activity at pH 8.0.
• Retains >80% of its activity at temperatures of 20–60°C
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