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

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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