Faculty of Health and Wellness Sciences. BHSc.

Medical Laboratory Science Molecular Notes DNA extraction

DNA EXTRACTION

Department of Biomedical Sciences 1 Cytogenetics 29th April 2013

http://www.enotes.com/dna-isolation-methods-reference/dna-isolation-methods Application: Isolation of DNA is needed for genetic analysis, which is used for scientific, medical, or forensic purposes. Scientists use DNA in a number of applications, such as introduction of DNA into cells and animals or plants, or for diagnostic purposes. In medicine the latter application is the most common. On the other hand, forensic science needs to recover DNA for identification of individuals (for example rapists, petty thieves, accident, or war victims), paternity determination, and plant or animal identification. Interfering substances: Presence of proteins, lipids, polysaccharides and some other organic or inorganic compounds in the DNA preparation can interfere with DNA analysis methods, especially with polymerase chain reaction (PCR). They can also reduce the quality of DNA leading to its shorter storage life. Common samples from which DNA can be extracted: Sources for DNA isolation are very diverse. Basically it can be isolated from any living or dead organism. Common sources for DNA isolation include whole blood, hair, sperm, bones, nails, tissues, blood stains, saliva, buccal (cheek) swabs, epithelial cells, urine, paper cards used for sample collection, bacteria, animal tissues, or plants. Principles in DNA extraction A number of commercial DNA purification kits use the same principles. Lysis solutions contain: sodium chloride; tromethamine (also known as Tris), which is a buffer to retain constant pH Ethylenediaminetetraacetic acid (EDTA), which binds metal ions Sodium dodecyl sulfate (SDS), which is a detergent. A common enzyme used in DNA extraction is Proteinase K. Collection of the isolated DNA The Basic steps in DNA extraction 1. Cell disruption or lysis to release the cellular DNA. This can be mechanical (sonicating or grinding), or by chemical means. 2. Membrane lipids are removed by detergents. 3. Enzyme removal of proteins using proteases. 4. Removal of RNA with RNase. 5. Precipitation of the DNA can be done with alcohol ethanol or isopropanol. (DNA is insoluble in these alcohols- DNA can be resolubilised in pure water or a slightly alkaline buffer) 6. The removal of Mg2+ and Ca2+ cations will prevent enzymes such as DNase from degrading the isolated DNA.

Faculty of Health and Wellness Sciences. BHSc. Medical Laboratory Science Molecular Notes DNA extraction

Department of Biomedical Sciences 2 Cytogenetics 29th April 2013

Quantity of DNA recovered A Diploid Cell contains approximately 6 pg of DNA Sperm contains approximately 3 pg of DNA The average WBC of an adult is 5 - 10 X 106 cells per ml of blood. Therefore, the theoretical recovery of DNA per ul of blood is 30 - 60 ng The RFLP procedure on requires a minimum of 50 ng of high molecular weight double stranded DNA. The PCR reactions call for on average 1 ng of DNA (single or double stranded). Quantitation of recovered DNA A tabletop spectrophotometer called a nanodrop uses 2ul for quantification. Nucleic acids absorb light at a wavelength of 260nm. When a 260 nm light source shines on a DNA sample, the light that passes through can be measured, and the amount of light absorbed can be measured. For double stranded DNA, an optical density (OD) of 1 at 260 nm correlates to a DNA concentration of 50ng/ul. Therefore the DNA concentration can be calculated from the optical density reading. NB A blank of purified water or the buffer without DNA should be used as an internal quality control measure ie to get a zero reading when there is no DNA present.

Methods: Organic (Phenol-Chloroform) Extraction Non-Organic (Proteinase K and Salting out) Chelex (Ion Exchange Resin) Extraction FTA Paper (Collection, Storage, and Isolation) Silica Based (Silica exchange resin- Qiagen) Magnetic Beads (see diagram on next page) The use of magnetic beads can be automated, increases throughput, can remove amplification inhibitors, uses no hazardous chemicals. The principle underlying magnetic bead procedures involves attracting DNA to magnetic beads, holding the beads in place using a magnetized source, such as a rack or tube holder, and washing away other components of the sample. Invitrogen's ChargeSwitch Technology (CST) involves the use of magnetic beads whose surface bears a charge that can be switched based on the pH of the surrounding buffer environment. At low pH, the beads are positively charged, attracting the negatively charged DNA molecules and allowing proteins and contaminants to be removed by washing. The ChargeSwitch Forensic DNA Purification Kit includes: Lysis buffer Magnetic beads Proteinase K Purification buffer Wash buffer Elution buffer

Faculty of Health and Wellness Sciences. BHSc. Medical Laboratory Science Molecular Notes DNA extraction

Department of Biomedical Sciences 3 Cytogenetics 29th April 2013