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Principles of Dna Isolation & Purification

DNA can be isolated from nucleated cells using a process that involves lysing cells, purifying the DNA from proteins, and precipitating the DNA out of solution using salt and alcohol. Key steps include lysing cells with detergents like SDS and enzymes like proteinase K to remove proteins, using phenol/chloroform extraction and salt precipitation to further purify the DNA from remaining cell debris and proteins, and finally precipitating the purified DNA out of solution with alcohol for collection and resuspension.

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326 views21 pages

Principles of Dna Isolation & Purification

DNA can be isolated from nucleated cells using a process that involves lysing cells, purifying the DNA from proteins, and precipitating the DNA out of solution using salt and alcohol. Key steps include lysing cells with detergents like SDS and enzymes like proteinase K to remove proteins, using phenol/chloroform extraction and salt precipitation to further purify the DNA from remaining cell debris and proteins, and finally precipitating the purified DNA out of solution with alcohol for collection and resuspension.

Uploaded by

ki_dvm
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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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

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