Molecular Biology Lab - 02 DNA RNA Extraction

MOLECULAR BIOLOGY AND DIAGNOSTICS LABORATORY DNA/RNA EXTRACTION OUTLINE • DNA & RNA Extraction o What is DNA & RNA? o Purpose of Extraction o Preparing Sample o Isolation of DNA o Isolation of RNA • DNA/RNA Extraction o Introduction o Principle of the Test o Materials o Procedure DNA AND RNA EXTRACTION (PPT) WHAT IS DNA & RNA? CENTRAL DOGMA OF MOLECULAR BIOLOGY PURPOSE OF EXTRACTION • • • • Difference between DNA & RNA DNA RNA Component Base + Sugar + Phosphate Common Adenine, Guanine, Cytosine Nitrogenous Base Unique Nitrogenous Thymine Uracil Base Sugar Ribose Deoxyribose Ribose PREPARING SAMPLE • • • • Chromosomes are derived from nucleated cells To release the nucleic acid from the cell o Unmask DNA from chromosome in nucleus The target nucleic acid should be free of contamination with protein, carbohydrate, lipids, or other nucleic acid o DNA should be purified from debris (protein, RNA,..) Example: o DNA free of RNA o RNA free of DNA Nucleated Cells in Suspension o Specimen may have to be pretreated to make nucleated cells available from which the nucleic acid will be extracted o Blood is preferred - easier to collect, ample amount o Example: ▪ WBC must be isolated from blood and bone marrow → differential density gradient centrifugation or differential lysis ▪ whole blood or bone marrow mixed with isotonic saline is overlaid with Ficoll. Tissue Samples o Grinding the frozen tissue in liquid nitrogen, homogenizing the tissue, or simply mincing the tissue using a scalpel can disrupt whole tissue samples. o Fixed embedded tissue has to be deparaffinized by soaking in xylene. o After xylene treatment, the tissue is usually rehydrated by soaking it in decreasing concentrations of ethanol. Microorganisms o Some bacteria and fungi have tough cell walls that must be broken to allow the release of nucleic acid o Enzyme products o Mechanically by grinding or by vigorously mixing with glass beads o o Treatment with detergent (1% sodium dodecyl sulfate) and strong base (0.2 M NaOH) in the presence of Tris base, ethylenediaminetetraacetic acid (EDTA), and glucose Boiling in 8% sucrose, 8% Triton X-100 detergent, Tris buffer, and EDTA after lysozyme treatment SOLID PHASE ISOLATION ISOLATION OF DNA • • • • Organic Isolation Methods Inorganic Isolation Methods Solid-Phase Isolation Crude Lysis ORGANIC ISOLATION METHOD • Lysis → Purification → Centrifugation → DNA collection → DNA Precipitation DNA / RNA EXTRACTION INTRODUCTION • INORGANIC ISOLATION • Lysis → Purification → Centrifugation → DNA collection → DNA Precipitation The basic criteria that any method of DNA isolation from any sample type should meet includes: efficient extraction of DNA from the sample , production of a sufficient amount of DNA for use in downstream processes, successful removal of contaminants, isolation of high quality and high purity DNA PRINCIPLE OF THE TEST • Cell lysis is required to make the DNA/RNA accessible. The purification of DNA and RNA molecules is required to avoid contaminations with other intracellular components, such as proteins and metabolites. MATERIALS • • SOLID PHASE ISOLATION Commercially available DNA/RNA Extraction and Purification o lysis buffer: 1% SDS, 0.5 M NaCl o isopropanol o 70% (v/v) ethanol2 Columns With Car o Collect the cells for DNA extraction into 2 ml tubes PROCEDURE DNA EXTRACTION • ISOLATION OF RNA • • Organic Isolation Methods Solid-Phase Isolation ORGANIC ISOLATION METHODS 1. 2. 3. 4. 5. 6. Note: If you are using cell suspensions you can move directly to the lysis procedure. If you are using samples, like cell tissues that are difficult to homogenize, you can use liquid nitrogen to create a homogeneous cell suspension. At the time you have a cell suspension, you should be careful about the membrane structure of your sample. Usually, for cells with a cell walls, it is recommended a pre-lysis treatment to degrade this barrier. You can use an enzymatic method or a mechanical method. Add 1ml of cell suspension into 2 ml tubes Add 100 mg of 0.5 mm glass beads and vortex for 15 min. Spin the cell suspension at top speed for 1 min using a centrifuge, Discard the supernatant and resuspend in 1000 μl of the lysis buffer. Spin at top speed for 1 min and Transfer the supernatant into a new 2ml tube. Add 500 μl of isopropanol and mix gently. 7. 8. 9. 10. 11. 12. 13. 14. Place the mixture on ice for 5 min. Spin at top speed 1min, Discard the supernatant and wash the DNA pellet with 500 μl 70% (v/v) ethanol. Spin again at top speed for 1 min. Discard the supernatant. Let the pellet air-dry, placing the 2 ml tubes upside down in a paper towel. Dissolve the DNA in 50 μl ultrapure DEPC H2 Use the Nanodrop equipment to access DNA concentration and quality. RNA EXTRACTION 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Spin the cell suspension at top speed for 1 min using a centrifuge, Discard the supernatant and resuspend in 1 ml of lysis buffer prewarmed at 65 ºC. Add 900 μl of acid phenol: chloroform and vortex for 10 s. Let the 2 ml tube on the bench for 10 min at room temperature. Spin at top speed for 10 min at 4 °C Transfer the supernatant to new 2 ml tube Add 0.3 volume of 5 M sodium acetate and 0.7 volume of acid phenol: chloroform. Mix gently the tube and incubate on ice for 10 min. Topspin for 10 min at 4 °C Transfer the supernatant to fresh 2 ml tubes. Add 0.1 volume of 3 M sodium acetate and the same volume of isopropanol. Incubate the tubes at -20 °C for 1 h. Spin at top speed for 10 min at 4 °C and discard the supernatant. 14. Wash the pellet with 500 μl ethanol (70 %) Centrifuge at 7,500 g for 5 min at 4 °C.10. Air-dry the pellet for 5–10 min 17. Dissolve in 25 μl RNase free water. •