DNA isolation.pptx

DNA EXTRACTION METHODS & ESTIMATION  What are theessential components of a DNA extraction Procedure? 1. Maximize DNA recovery 2. Remove inhibitors 3. Remove or inhibit nucleases 4. Maximize the quality of DNA  How Much DNA Can We Recover? A Diploid Cel...

DNA EXTRACTION METHODS & ESTIMATION  What are theessential components of a DNA extraction Procedure? 1. Maximize DNA recovery 2. Remove inhibitors 3. Remove or inhibit nucleases 4. Maximize the quality of DNA  How Much DNA Can We Recover? A Diploid Cell contains approximately 6 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. How MuchDNA Do We Need? The PCR reactions call for on average 1 ng of DNA (single or double stranded). Many of the commercially available kits are sensitive below 1 ng of DNA (100-250 pg).  Basic steps for DNA 1.extraction Breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by grinding, sonicating or treating the sample with lysis buffer. 2. Removing membrane lipids by adding a detergent. Purposes of the Extraction Buffer Detergents Chaotropic 1. Dissolve cellular salts CTAB membranes Detergents Metal chelators 2. Inactivation of DNase and Reducing Rnase agents Salt s 3. Assist in the removal of CTAB contaminants PVP  Extraction/Precipitation Method Use of Detergents to Lyse Cells: Mixed micelle Plasma membrane (phospholipid bilayer) Detergent molecules + SDS 3. Removing proteins by adding a protease (optional but almost always done). 4.Precipitating the DNA with an alcohol — usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt.  Depending on the material and requirement the extraction process can be modified with alteration of chemicals used  Extraction/Precipitation Method Step 1: Disruption of cell walls by grinding Step 1+2: mechanical disruption and homogenization in extraction buffer Grind sample into a fine powder to shear cell walls and membranes Step 2: Lysis of cells in extraction buffer A homogenizer allows cells to be mechanically disrupted within the extraction buffer Mix thoroughly with extraction buffer to dissolve cell membranes and inhibit Crude nuclease activity lysate  Figure 5.5 Tissue disruption using a Figure 5.4 An automated pressure-generating instrument also mechanical disruption device known as a barocycler. for efficient disruption of During the process, a barocycler is tissues. It can utilized to apply alternating cycles of be used for high-throughput high and low pressures onto applications involving sample specimens placed in single-use preparation for the isolation of processing containers. The technique nucleic is known as pressure-cycling acids. (c Richard C. Li.) technology (PCT). (c Richard C. Li.) Lysis of Cellular and Organelle Membranes During or after tissue disruption, membranes—including those of cells, nuclei, and mitochondria—are lysed in order to release DNAs, including nuclear and mitochondrial DNA. The lysis can be carried out using salts and chaotropic agents (Section 5.2.3) such as guanidinium salts and detergents such as sarkosyl and sodium dodecyl sulfate (SDS). These substances can destroy membranes, denature proteins, and dissociate proteins such as histones from DNA. The lysis procedureis usually carried out in a buffer, such as Tris, in order to maintain a pH where endogenous deoxyribonucleases (DNases) remain inactive. DNases are a type of nuclease that catalyzes the cleavage of phosphodiester bonds of DNA. Endogenous DNases are located in cytoplasmic lysosomes and play a role in degrading the DNA of invading viruses. When cells are lysed, the DNases that are also released can degrade the extracted DNA. Chelating agents such as EDTA or ChelexR can therefore be used to chelate the divalent cations that are the cofactors of DNases, in order to inhibit DNase activities.  Most Commonly used DNA Extraction Procedures Organic (Phenol-Chloroform) Extraction Non-Organic (Proteinase K and Salting out) NCM / Nylon membrane(Collection, Storage, and Isolation) The method utilized may be sample dependant, technique dependant, or analyst preference 5.2 Methods of DNA Extraction 5.2.1 Extraction with Phenol–Chloroform This method is also called organic extraction. Major steps include the following: 5.2.1.1 Cell Lysis and Protein Digestion These steps can be achieved by digestion with proteolytic enzymes such as proteinase K before extraction with organic solvents. 5.2.1.2 Extraction with Organic Solvents The removal of proteins is carried out by extracting aqueous solutions containing DNA with a mixture of phenol:chloroform:isoamyl alcohol (25:24:1). Phenol is used to extract the proteins from the aqueous solution. Although phenol has a slightly higher density than water, it is sometimes difficult to separate it from the aqueous phase. Therefore, chloroform is utilized as it has a higher density than phenol. As a result, the phenol–chloroform mixture forms the organic phase at the bottom of the tube and is easily separated from the aqueous phase. Isoamyl alcohol 5.2.1.3 Concentrating DNA Two common methods for concentrating DNA are ethanol precipitation and ultrafiltration. In the first method, the DNA is precipitated from the aqueous solution with ethanol and salts. Ethanol depletes the hydration shell of DNA, thus exposing its negatively charged phosphate groups.  Extraction/Precipitation Step 3: Organic Method extraction Mix thoroughly Aqueou s with an equal volume of Centrifug Collect aqueous phase organic solvent e Interphas e.g. phenol, e chloroform, or Organi phenol:chloroform c Perform additional extractions for increased purity Crude lysate The aqueous phase contains containing nucleic water- soluble molecules, acids and other cell including nucleic acids. Proteins constituents and lipids become trapped in the organic phase, and are thus separated away. Insoluble plant debris become trapped in the interphase between the two layers  Extraction/Precipitation Method Step 4: Nucleic Acid Precipitation Before After Supernata 70% nt EtOH Centrifug Was Centrifug e h e Pelle t Dissolve pellet (H2O, Add alcohol and salt Pellet Pelletdown downnucleic nucleicacids. TE, etc.) to precipitate nucleic acids. Wash pellet with 70% ethanol to acids from the remove aqueous fraction residual salts and other contaminants. Discard ethanol and allow pellet to dry.  EXTRACTION Perhaps the most basic of all procedures in genetic engineering is the purification of DNA. The key step, the removal of proteins, can often be carried out simply by extracting aqueous solutions of nucleic acids with phenol and/or chloroform.  EXTRACTION Cell Lysis Buffer – This buffer will lyse cell membrane, nuclei are intact, pellet nuclei. The nuclei is Resuspended in a buffer containing Sodium Dodecly Sulfate (SDS) and Proteinase K. This will Lyse nuclear membrane and digest protein. DNA released into solution is extracted with phenol-chloroform to remove proteinaceous material.  ORGANIC EXTRACTION REAGENTS Cell Lysis Buffer - Non-ionic detergent , Salt, Buffer, EDTA designed to lyse outer cell membrane, but will not break down nuclear membrane. EDTA (Ethylenediaminetetraacetic disodium salt) is a chelating agent of divalent cations such as Mg2+. Mg2+is a cofactor for Dnase nucleases. If the Mg2+is bound up by EDTA, nucleases are inactivated.  ORGANIC EXTRACTION REAGENTS Proteinase K - it is usual to remove most of the protein by digesting with proteolytic enzymes such as proteinase K, which are active against a broad spectrum of native proteins, before extracting with organic solvents. Protienase K is approximately 10 fold more active on denatured protein. Proteins can be denatured by SDS or by heat.  ORGANIC EXTRACTION REAGENTS Phenol/Chlorform - The standard way to remove proteins from nucleic acids solutions is to extract once with phenol, once with a 1:1 mixture of phenol and chloroform, and once with chloroform. This procedure takes advantage of the fact that deproteinization is more efficient when two different organic solvents are used instead of one. Also, the final extraction with chloroform removes any lingering traces of phenol from the nucleic acid  ORGANIC EXTRACTION REAGENTS Phenol - often means phenol equilibrated with buffer (such as TE) and containing 0.1% hydroxyquinoline and 0.2% b-ercaptoethanol (added as antioxidants. The hydoxquinoline also gives the phenol a yellow color,making it easier to identify the phases (layers). Chloroform - often means a 24:1 (v/v) mixture of chloroform and isoamyl alcohol. The isoamyl alcohol is added to help prevent foaming.  Concentrating DNA by Alcohol Precipitation The most widely used method for concentrating DNA is precipitation with ethanol. The precipitate of nucleic acid, forms in the presence of moderate concentrations of monovalent cations (Salt, such as Na+), is recovered by centrifugation and redissolved in an appropriate buffer such as TE. The technique is rapid and  phenol helps to remove non polar proteins and lipids from the solution, phenol is used to denature the proteins. ethanol change ionic potential of DNA and remove water molecules, which help in precipitation of DNA. Salt would attract the phosphate ends of DNA, therefore it pulls it way from other substances in the sample (Separation of DNA from surroundings)  Concentrating DNA Alcohol Precipitation The four critical variables are the purity of the DNA, its molecular weight, its concentration, and the speed at which it is pelleted. DNA a concentrations as low as 20 ng/ ml will form a precipitate that can be quantitatively recovered. Typically 2 volumes of ice cold ethanol are added to precipitate  Concentrating DNA Alcohol Precipitation Very short DNA molecules (