DIAGNOSTICS MOLECULAR BIOLOGY

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1 1 DIAGNOSTICS MOLECULAR BIOLOGY MLSC 4050 Professor Labiner Module 1 Lecture 4: Nucleic Acid Extraction

2 Nucleic Acid Extraction: Objectives 2 Students will be able to: 1. List the types of specimens that are acceptable for isolating nucleic acid. 2. Compare and contrast organic, inorganic, and solid-phase approaches for isolating cellular DNA. 3. Note the chemical conditions in which DNA precipitates and goes into solution. 4. State the purpose of each reagent used in DNA isolation procedures. 5. Describe the gel-based, spectrophotometric, and fluorometric methods used to determine the quantity and quality of DNA and RNA preparations. 6. Calculate the concentration and yield of DNA and RNA from a given nucleic acid preparation. 7. Distinguish between the isolation of total RNA with that of messenger RNA.

3 Purpose for Extraction 3 Nucleic acids released from cell Need pure target (DNA or RNA) Remove protein, carbohydrates & lipids Isolate nucleated cells Cell Lysis Purification Confirm Purity and Concentration

4 Specimens Expected Yield Blood* Buffy coat Bone marrow Solid tissue Lavage fluids Bacteria, Viruses & Fungi Mitochondria (20 50µg ) (50 200µg) ( µg) (1 10µg) (2 250µg) (10µg 1mg) (2 10 µg) * DNA from Whole Blood is obtained from White Blood Cells since mature Red Blood Cells have no nucleus

5 Specimens: Adequate for amplification by Polymerase Chain Reaction (PCR) Specimens Expected Yield Dried blood Buccal cells Saliva Bone, teeth Hair follicles Cerebrospinal fluid Fixed tissue ( µg) (1 10µg) (5 15µg DNA/ml) (30 50 µg) ( µg) (0.3 3 µg) (6 50 µg)

6 Choosing an Isolation Method 6 Important considerations: Processing speed Ease of use Yield of DNA or RNA Quality of DNA and RNA prepared Purity of DNA Quality assurance criteria Cost of preparation

7 DNA Extraction Methods 7 Organic: uses organic chemicals, phenol, chloroform Inorganic: uses inorganic chemicals, detergents, ethylenediamine tetraacetic acid (EDTA), acetic acid, salt (salting out) Low ph & high salt conditions Solid phase: DNA is immobilized on a solid support, beads, or columns

8 Organic DNA Isolation 8 1 Lysis (NaOH, SDS) 2 Acidification (acetic acid, salt ) 4 DNA Precipitation (ethanol) 3 Extraction (phenol, chloroform)

9 Inorganic DNA Isolation 9 Lysis (Tris,EDTA,SDS) 3 1 DNA Precipitation (isopropanol) 2 Protein Precipitation (sodium acetate)

10 Solid Phase DNA Isolation 1 2 Lysis (supplied reagents) Acidification (supplied reagents) 5 Elute DNA (low salt) 4 Wash (supplied buffer) 3 Adsorption (low ph) 10

11 11

12 12 DNA Isolation Method Comparison Liquid Phase (Lyse RBCs) Lyse cells (Protein digestion-prok) Separate proteins from DNA Precipitate DNA-alcohol Solid Phase (Pre-lyse cells) Apply sample Wash Elute DNA Rehydrate DNA

13 Method Comparison 13 Organic Limited sample Slow, labor-intensive Toxic (phenol/chloroform), fume hood required, disposal of hazardous materials required Inorganic Fast and easy method Nontoxic materials, no fume hood required, no hazardous materials disposal issues, Produces high-quality DNA Solid Phase Fast and easy No precipitation required

14 Measurement of Nucleic Acid 14 Minimizes variability Methods for assessing quantity, quality, and molecular size Agarose gel electrophoresis Fluorometry Spectrophotometry Genomic DNA should look like this

15 Methods to Assess DNA 15 Spectrophotometry 1 OD 260 = 50μg/ml dsdna - Concentration = 40μg/ml RNA μg/ml x ml = μg DNA -Yield OD 260 / OD 280 -Measure of purity OD 260 / OD 280 ~ ; Good DNA! < 1.6; too much protein or contaminates

16 Quantity from UV Spectrophotometry 16 [DNA] = (A 260 A 320 ) x dilution factor x 50 µg/ml [RNA] = (A 260 A 320 ) x dilution factor x 40 µg/ml OD 260 measures nucleic acid OD 280 measures protein OD 320 background scatter Concentration = µg of DNA or RNA per ml of hydrating solution

17 Manual Yield Calculation 17 Example of manual quantitation calculation if you made a 1:20 dilution: A 260 = 0.20 absorbance units 0.2 x 20 (dilution) x 50 µg /ml = 200 µg /ml Sample volume = 0.2 ml 200 µg /ml x 0.2 ml = 40 µg

18 Types of RNA 18 Messenger RNA (mrna) Ribosomal RNA (rrna) Transfer RNA (trna) Heteronuclear RNA (hnrna) Small nuclear RNA (snrna) Many small/micro RNAs 5 3 A A A A A A A A A T T T T T T T T T 3 5 Bead or column

DNA-RNA EXTRACTION. Dr. Amira A. T. AL-Hosary Lecturer of infectious diseases, Faculty of Veterinary Medicine, Assiut University, Egypt

DNA-RNA EXTRACTION. Dr. Amira A. T. AL-Hosary Lecturer of infectious diseases, Faculty of Veterinary Medicine, Assiut University, Egypt DNA-RNA EXTRACTION Dr. Amira A. T. AL-Hosary Lecturer of infectious diseases, Faculty of Veterinary Medicine, Assiut University, Egypt Nucleic Acids (DNA & RNA) DNA and RNA Breaks (Nucleotides) I. DNA