DNA Extraction And PurificationDNA Extraction And Purification

BYBY

Dr. Naglaa FathyDr. Naglaa Fathy Lecturer of Biochemistry and Molecular Lecturer of Biochemistry and Molecular

Biology , faculty of medicine ,Biology , faculty of medicine ,

Benha universityBenha university

2008 2008

Structure of the cellStructure of the cell

• Plasma membrane and membranes of organelles nuclear envelope included• DNA located in nucleus

• A lot of proteins around

• Mitochondrial DNA

Extraction of genomic DNAExtraction of genomic DNA

Cell collectionCell collection Add Lysis buffer to cells to break open Add Lysis buffer to cells to break open

cell and nuclear membranes and release cell and nuclear membranes and release nuclear contentsnuclear contents

Digest sample with protease to degrade Digest sample with protease to degrade proteinsproteins

Precipitate DNA with cold alcohol in high Precipitate DNA with cold alcohol in high saltsalt

Lysis bufferLysis buffer

Lysis bufferLysis buffer 50 mM Tris-HCI, pH 8.0 to 50 mM Tris-HCI, pH 8.0 to

maintain the pH of the solution at maintain the pH of the solution at a level where DNA is stablea level where DNA is stable

1% SDS to break open the cell 1% SDS to break open the cell and nuclear membranes, allowing and nuclear membranes, allowing the DNA to be released into the the DNA to be released into the solution (SDS also denatures and solution (SDS also denatures and unfolds proteins,making them unfolds proteins,making them more susceptible to protease more susceptible to protease cleavage)cleavage)

Why add protease?Why add protease?

Protease destroys nuclear proteins that Protease destroys nuclear proteins that bind DNA and cytoplasmic enzymes that bind DNA and cytoplasmic enzymes that breakdown and destroy DNAbreakdown and destroy DNA

Protease treatment increases the amount Protease treatment increases the amount of intact DNA that is extractedof intact DNA that is extracted

Adding saltAdding salt

• • The addition of NaCI allows theThe addition of NaCI allows the DNA molecules to come togetherDNA molecules to come together instead of repelling each other,instead of repelling each other, thus making it easier for DNA tothus making it easier for DNA to precipitate out of solution whenprecipitate out of solution when alcohol is addedalcohol is added • • Na+ ions bind to the phosphateNa+ ions bind to the phosphate groups of DNA molecules,groups of DNA molecules, neutralizing the electric charge ofneutralizing the electric charge of the DNA moleculesthe DNA molecules • • Our protease solution alreadyOur protease solution already contains saltcontains salt

Precipitation of DNAPrecipitation of DNA DNA does not dissolve in alcohol.DNA does not dissolve in alcohol. Addition of cold alcohol makes the DNA clump Addition of cold alcohol makes the DNA clump

together and precipitate out of solutiontogether and precipitate out of solution

Precipitated DNA molecules appear as long Precipitated DNA molecules appear as long pieces of fluffy, stringy, web-like strands.pieces of fluffy, stringy, web-like strands.

Microscopic oxygen bubbles Microscopic oxygen bubbles “aggregate”together, as the DNA precipitates. “aggregate”together, as the DNA precipitates.

The larger, visible air bubbles “lift” the DNA The larger, visible air bubbles “lift” the DNA out of solution, from the aqueous into the out of solution, from the aqueous into the organic phaseorganic phase

The DNA in the glass vial can last foryearsThe DNA in the glass vial can last foryears

DNA Extraction & Purification:DNA Extraction & Purification: Key Steps Key Steps

Lysis of the cellsLysis of the cells Removal of contaminantsRemoval of contaminants

ProteinsProteins

RNARNA

Other macromoleculesOther macromoleculesConcentration of purified DNA (if Concentration of purified DNA (if

required)required)

Standard Protocol for DNA Standard Protocol for DNA ExtractionExtraction

Organic solventOrganic solvent Salting outSalting out Cation Exchange ResinsCation Exchange Resins

The Standard Method-1The Standard Method-1

Lysis of cells:Lysis of cells:

Lysis buffer: SDS and/or 8.0 M ureaLysis buffer: SDS and/or 8.0 M urea Removal of contaminantsRemoval of contaminants::

Proteinase KProteinase K

Phenol:chloroform extractionPhenol:chloroform extraction Concentration of DNA:Concentration of DNA:

Ethanol precipitationEthanol precipitation

Phenol: chloroform: isoamyl Phenol: chloroform: isoamyl alcohol mixture:alcohol mixture:

Phenol :Phenol : - Denatures proteins - Denatures proteins - Solubilizes denatured proteins- Solubilizes denatured proteins

ChloroformChloroform -Denatures proteins -Denatures proteins -Stabilizes the aqueous/organic -Stabilizes the aqueous/organic boundaryboundary

Alcohol Alcohol -Separation of aqueous/organic -Separation of aqueous/organic - phases Prevents foaming upon - phases Prevents foaming upon vortex vortex

DNA Extraction & Purification:DNA Extraction & Purification:The Standard Method-3The Standard Method-3

Tris-buffered, redistilled phenol, pH 8.0: Oxidation products can damage DNA chains Addition of 8-hydroxyquinolone:-antioxidant

-yellow color of phenol

DNA is found in the upper aqueous layer

Exceptions: high salt content, the aqueous phase forms lower layer

If phenol is not equilibrated, DNA in the organic phase

DNA Extraction & DNA Extraction & Purification:Purification:

The Standard Method-4The Standard Method-4 Ethanol precipitation:

Concentration DNA solutionsRemoval of residual organic solventsDNA-free solutes

Ethanol induces a structural transition in DNA precipitation High monovalent cation conc. (0.1 – 0.5 M)

Most salts are soluble in 70% ethanol, precipitationand washing desalt DNA

DNA Extraction & DNA Extraction & Purification:Purification:

The Standard Method-5The Standard Method-5

Time-consuming

Hazardous organic solvents

Residual amounts of organic solvents interfere with accurate measurement of DNA conc. with enzymatic manipulations of DNA

Disadvantages:

DNA Extraction & DNA Extraction & Purification:Purification:

The Standard Vs other The Standard Vs other MethodsMethods

Key Differences:

Sample: Type Quantity/volume

Requirement:Time for extractionEquipments and reagentsCost/sample

DNA: Yield & conc.Purity

DNA Extraction & DNA Extraction & Purification:Purification:

QIA amp DNA mini kits-1QIA amp DNA mini kits-1(1)(1) Lysis of cellsLysis of cells

(2) Spin column: DNA adsorption to (2) Spin column: DNA adsorption to silica-gel membranesilica-gel membrane

(3) Two-different wash steps: Removal of (3) Two-different wash steps: Removal of contaminantscontaminants

(4) Elution: Pure and concentrated DNA(4) Elution: Pure and concentrated DNA

DNA Extraction & DNA Extraction & Purification:Purification:

QIA amp DNA mini kits-2QIA amp DNA mini kits-2AdvantagesAdvantages

•Simple and rapid• No use of organic solvents• Sample: versatile• Pure and concentrated DNA

DNA Extraction & DNA Extraction & Purification:Purification:EvaluationEvaluation

DNA purity: A260/A280 ratioDNA purity: A260/A280 ratio

1.7 – 1.91.7 – 1.9

DNA concentration (μg/ml):DNA concentration (μg/ml):

A260 X 50A260 X 50 DNA yield:DNA yield:

DNA conc. X Total volume of DNA DNA conc. X Total volume of DNA solutionsolution