DNA extraction

DNA is building block of the life for all living creatures. Every thing from bacteria to human has DNA in their cellular stucture. Every thing living contain DNA DNA extraction is a routine procedure to collect DNA for subsequent molecular or forensic analysis.

Structure of the cell

Physical Characteristics of DNA DNA absorbs UV light at 260 nm Allows quantitation DNA is water soluble DNA precipitates in alcohols DNA carries a net negative charge DNA has characteristic melting & annealing temperatures

Why extract DNA? The isolation of the DNA from biological sample is an essential step in the DNA technology (PCR - RFLP- cloning - hyberdization all this approaches require DNA as template • disease diagnosis • DNA sequencing • genetically modified organisms (GMO) - agriculture, pharmaceutical

Nucleic Acid Preparation Sample Source? Amniocytes or amniotic fluid Dried blood spots Fresh or frozen tissue (biopsy material) Sputum, urine, CSF, or other body fluids Fixed or paraffin-embedded tissue Whole blood Buffy coat Serum or plasma Bone material Buccal cells Cultured cells

How to isolate DNA? They are many types of methods to isolate DNA depending on the type of the sample and the purpose from extraction (organic method (phenol – chloroform )-chlex –methanol - commercial kit (FTA) In genral all type of methods are following three coral steps 1-lyses cell wall by lyseis buffer 2-precipitation of proteins 3- precipitation of DNA

1-Breaking the cells open, commonly referred to as cell disruption, to expose the DNA within. 2-Removing membrane lipids by adding a detergent.

Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents • 50 mM Tris-HCI, pH 8.0 to maintain the pH of the solution at a level where DNA is stable 1% SDS to break open the cell and nuclear membranes, allowing the DNA to be released into the solution (SDS also denatures and unfolds proteins, making them more susceptible to protease cleavage)

Precipitating the DNA with an alcohol — usually ethanol or isopropanol Precipitating the DNA with an alcohol — usually ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation.

DNA extraction – the basic concept Process Common procedure Cell lysis Protein removal DNA precipitation SDS, CTAB Proteinase K Freezing Grinding Chemical Enzymatic Mechanic Phenol chloroform Sodium chloride Sodium acetate Membrane Beads Organic solvents Salt DNA binding Alcohol Ethanol Iso-propanol

Cells Extract HOW? Organic extraction Brown p.28 Pure DNA

Phenol extraction of DNA samples Phenol extraction is a common technique used to purify a DNA sample

DNA Isolation Methods Liquid Phase Organic Extraction Phenol :chloroform/isoamyl alcohol Since phenol and water are immiscible, two phases form - a water phase and a phenol phase. The phases are then mixed thoroughly. This forces the phenol into the water layer where it forms an emulsion of droplets throughout. The proteins in the water phase are denatured and partition into the phenol, while the DNA stays in the water.

The mixture is then centrifuged and the phases separate. The DNA-containing water phase can now be pipetted off, and the phenol/protein solution is discarded.

Ethanol precipitation

Disadvantages: Time-consuming Hazardous organic solvents Residual amounts of organic solvents interfere

DNA extraction kit 􀂄 Two main advantages: Saves time and makes the process of DNA purification a relatively easy and straightforward process. Can handle up to 100 μg of DNA

Chelex Extraction Method • More rapid than organic extraction method • Involves few steps and fewer opportunities for contamination

Chelex extraction Put sample in tube Add 5% Chelex® beads; vortex Boil at 100°C Supernatant can be used directly for quantitation/PCR

Evaluation of Nucleic Acids Spectrophotometrically • quantity • quality

Assessment of DNA quality • gel electrophoresis Assessment of DNA quality