Molecular - Chapter 4 - Nucleic Acid Extraction Questions and Answers

Molecular - Chapter 4 - Nucleic Acid Extraction Questions and Answers Purpose of nucleic acid extraction to release nucleic acid (DNA or RNA) from the cell to use for subsequent procedures by lysing cell without damaging nucleic acid Target should be free from? contamination (protein, lipid, other nucleic acid) Previous Play Next Rewind 10 seconds Move forward 10 seconds Unmute 0:00 / 0:15 Full screen Brainpower Read More Methods for DNA isolation 1- organic, 2- inorganic, and 3- solid-phase Methods for RNA isolation 1-organic and 2-solid-phase Steps of Nucleic Acid Extraction 1-Lyse the cell to Release cellular material; 2-Purify target nucleic acid; 3-Determine concentration and purity why determining purity? Quality control before performing the test. Can be analyzed without DNA amplification and why? Blood Buffy coat Bone marrow Solid tissue Lavage fluids Bacterial cultures Fungi Organelles, mitochondria. Because quantity is sufficient. What samples are adequate for amplification by PCR? Dried blood; Saliva; Bone/teeth; Amniotic fluid; Hair follicles/hair shafts; Buccal cells; Cerebrospinal fluids; Fixed tissue; Feces; Soil. Mitochondrial DNA is different from? genomic DNA Best fixed DNA specimens Acetone; 10% buffered neutral formalin. Not as good fixed DNA specimens Zambonis, Clarkes Paraformaldehyde Formalin-alcohol-acetic acid Metharcan Less desirable fixed DNA specimens Carnoys, Zenkers, Bouins, B-5 Early DNA Extraction Methods Density gradient centrifugation Human DNA first isolated by Miescher in 1869 Organic dna extraction uses organic chemicals, phenol, chloroform (dissolve hydrophobic contaminants) Organic chemicals for dna extraction Phenol; Chloroform. Inorganic dna extraction uses inorganic chemicals, detergents, ethylenediamine tetraacetic acid (EDTA), acetic acid, salt (salting out, spooling) Inorganic chemicals for dna extraction detergents; ethylenediamine tetraacetic acid (EDTA); acetic acid; salt (salting out, spooling) Solid phase dna extraction DNA is immobilized on a solid support, beads, or columns solid phase for dna extraction Solid support; Beads; Columns. Specimens that need preparation before dna extraction Bone marrow; Peripheral blood; Tissue; Plants/fungi. Preparation of bone marrow and peripheral blood (3 techniques) 1- Density gradient centrifugation; 2- Differential osmolysis; 3- Streptokinase. Preparation of tissue (3 techniques). More involved before starting extraction. 1- Freeze/crush; 2- Mince; 3- Enzymatic digestion. Preparation of plant and fungi (3 techniques) 1- Homogenize; 2- Enzymes (lyzozyme or zymolyase); 3- Vortex with glass beads. Enzymes that digest cell wall polymers and are used for treatment of plant and fungi Lyzozyme; Zymolyase Steps of organic dna isolation 1- Lysis; 2- Acidification; 3- Extraction; 4- Precipitation organic dna - lysis NaOH; SDS organic dna - acidification Acetic acid; salt after acidification with acetic acid, dna remains in which phase? in the aqueous solution organic dna - extraction Phenol; Chloroform. organic dna - precipitation ethanol inorganic dna isolation steps 1- lysis; 2- protein precipitation; 3- DNA precipitation. low pH and high salt concentration inorganic DNA isolation inorganic dna - lysis Tris, EDTA, SDS inorganic dna - protein precipitation sodium acetate inorganic dna - DNA precipitation isopropanol solid-phase dna isolation steps 1- lysis; 2- acidifcation; 3-adsorption; 4-wash; 5-elute DNA SP dna - lysis supplied reagents (1) SP dna - acidification supplied reagents (2) SP dna - adsorption low pH SP dna - wash supplied BUFFER - to remove protein impurities SP dna - elution low SALT Solid-phase isolation media include: Slica spin columns; Silica beads.