MOLECULAR BIOLOGY Lap2: DNA Extraction 2/15/2019 Biotechnology College /Al-Qadisiyah University Molecular biology-second stage (oct.-2016). Alaa Kamil Abdulla

DNA extraction 2/15/2019 DNA extraction is a routine procedure used to isolate DNA from the nucleus of cells. Scientists can buy ready‐to‐use DNA extraction kits. These kits help extract DNA from particular cell types or sample types. However, they can be expensive to use routinely, so many labs have their own methods for DNA extraction.

DNA extraction Many different methods and technologies are available for the isolation of genomic DNA. All methods involve: Disruption and lyses of the starting material followed by removal of proteins and other contaminants and finally recovery of the DNA

Methods of DNA extraction 2/15/2019 1- Ethanol precipitation  Usually by ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. Precipitation of DNA is improved by increasing of ionic strength, usually by adding sodium acetate.

Methods of DNA extraction 2- Phenol–chloroform extraction   The  phenol which denatures a proteins in the sample. After centrifugation of the sample, denaturated proteins stay in the organic phase while aqueous phase containing nucleic acid is mixed with the chloroform that removes phenol residues from solution. 2/15/2019

Methods of DNA extraction 3- Minicolumn purification   That relies on the fact that the nucleic acids may bind to the solid phase (silica or other) depending on the pH and the salt concentration of the buffer. 2/15/2019

DNA extraction To choice of a method depends on many factors: A. The quantity and molecular weight of the DNA B. The purity required for application C. The time and expense

Sample Collection A- Source: Sample can be isolated from any living or dead organism Common sources for DNA isolation include: Whole blood Buffy coat Bone material Buccal cells Cultured cells Amniocytes or amniotic fluid Sputum, urine, CSF, or other body fluids

Sample Collection B. Sample age: May be fresh or has been stored . Stored sample can come from: Archived tissue samples , Frozen blood or tissue (biopsy material) , Exhumed bones or tissues & Ancient human sample. Dried blood spots

Extraction of DNA Key Steps Lyses of the cells Removal of contaminants includes Proteins RNA Other macromolecules Concentration of purified DNA

Step 1. Breaking cells open to release the DNA 2/15/2019 Put into a solution containing salt. The positively charged sodium ions in the salt help protect the negatively charged phosphate groups that run along the backbone of the DNA. A detergent is then added to breaks down the lipids in the cell membrane and nuclei. DNA is released as these membranes are disrupted.

The cells in a sample are separated from each other, often by a physical means such as grinding or vortexing. 2/15/2019 Homogenizers pestle in a microc- entrifuge tube Mortar and Pestle

Step 2. Separating DNA from proteins and other cellular debris 2/15/2019 To get a clean sample of DNA, it’s necessary to remove as much of the cellular debris as possible, by a variety of methods. Protease ( protein enzyme) is added to degrade DNA associated proteins and other cellular proteins. Some of the cellular debris can be removed by filtering the sample.

Step 3. Precipitating the DNA with an alcohol 2/15/2019 Finally, ice‐cold alcohol (either ethanol or isopropanol) is carefully added to the DNA sample. The DNA is soluble in water but insoluble in the presence of salt and alcohol. By gently stirring the alcohol layer with a sterile pipette, a precipitate becomes visible and can be spooled out. If there is lots of DNA, you may see a stringy, white precipitate.

Step 4. Cleaning the DNA 2/15/2019 The DNA sample can now be further purified (cleaned). It is then resuspended in a slightly alkaline buffer to cleaning up DNA from aqueous solutions to remove buffer salts, enzymes or other substances that could affect on other applications.

Step 5. Confirming the presence and quality of the DNA 2/15/2019 For further lab work, it is important to know the concentration and quality of the DNA. Optical density readings taken by a spectrophotometer can be used to determine the concentration and purity of DNA in a sample. Alternatively, gel electrophoresis can be used to show the presence of DNA in your sample and give an indication of its quality.