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Nucleic acids


The ideal technique
 
  • SENSITIVE 100% (no false negative) (it detects "all")
  • SPECIFIC 100% (no false positive) (it detects "only")
  • Safe (no dangerous or toxic compounds)
  • Inexpensive (cheap reagents and instruments)
  • Fast (man-hour)
  • Straightforward (easy to be performed)
  • Low intra- and inter-observer variability
    •  
    NUCLEIC ACIDS EXTRACTION METHODS

    Methods - Links to methodology websites in Molecular Biology - Work in Progress

    Molecular Cloning: A Laboratory Manual
    The first one-volume edition (1982) was authored by the molecular biologist Tom Maniatis,
    it has been known     for many years as "The Maniatis" and has been present
    in virtually any laboratory of Molecular Biology and Genetics.
    Subsequent three-volume editions were authored by Sambrook, Fritsch and Maniatis (1989)
    and by Sambrook and Russell (2001).
    Currently, the     fourth edition is available (Green and Sambrook, 2012)
    at the Cold Spring Harbor Laboratory (CSHL) publisher.

    Current Protocols in Molecular Biology (popularly referred to as "The Red Book")
    by Ausubel, Brent, Kingston, Moore, Seidman, Smith and Struhl
    consists of three looseleaf volumes and has been published by Wiley in 1988.
    Quarterly updates can be filed into the looseleaf.

    1) No extraction
    2) Extraction (isolation)

    Application:

    Cell type, tissue, species, etc
    1) Human tissues
    2) Organisms used for human gene cloning

    Quantity of the yielded material
     
    Quality of the yielded material
    * absence of degradation
      (method-induced)
    * absence of contaminants

    Functionality         of the yielded material
     

    Nucleic acid extraction phases

    Relevance of the knowledge of rational basis of the methods employed.
    Cromo (Chromium) - a story by Primo Levi ("Il sistema periodico", Einaudi, 2014).


    Cell separation
     
    Cell lysis (detergents; enzymes; chaotropic agents) - [RNase inhibition]

    DNA may then be yielded from the lysate according to three fundamental ways:
     

    1) Organic solvents

    Extraction (apolar solvent)
    Precipitation
        Salts (cations)
        Solvent (moderately apolar solvent)
        Temperature
        Gravity        
        Carrier
    Washing
    Suspension

    Examples using home-made reagents:
    Extracting DNA from fruit
    http://teach.genetics.utah.edu/content/labs/DNA_Extraction.pdf
    Simulator:
    http://learn.genetics.utah.edu/content/labs/extraction/


    2) Affinity (glass beads)

    In presence of high concentration of chaotropic salts (NaI, GuSCN), nucleic acids bind to glass particles or silica gels.
    Nucleic acid elution is obtained through low-salt concentration solutions.
     

    3) Ionic exchange

    Under a certain salt concentration, a positively charged chromatographic matrix retains negatively charged nucleic acids molecules.
    (e.g., Qiagen resin for plasmid DNA extraction).


    Nuclec acid extraction check


    1. Quality control

    Gel electrophoresis

    Simulator
    Agarose vs Acrylamide. Concept - Molecule. Vertical casting.
    Run homogeneity. Example cells: Owl B3 - Hoefer Super-Sub He100

    Electrophoresis: Notable numbers


    • Nucleic acid presence.
    • Nucleic acid size: DNA, >20 kb   |   RNA: 28S and 18S bands.
    • Nucleic acid integrity (absence of degradation).


    Examples: DNA (1); RNA (1); RNA (2)

    Spectrophotometer
    260 nm/280 nm: 1.8


    2. Quantity estimation

    Gel electrophoresis
  • Estimation relative to a Marker

    • Spectrophotometer
  • Estimation by absorbance


    • 3. Functionality check
     
    Enzymatic reactions (e.g, digestion)
     

    DNA and RNA extraction organization

    Data recording
    Bank
    Conservation

    Diagnostics
    (genomic, mitochondrial, viral)

    Saliva
    Blood
    Body fluids
    Swab - Buccal, nasal, pharyngeal, ocular...
    Tissues
    Tissue slides
    Hair
    Sperm

    Legal issues - informed consent (example)
     
    Useful numbers

    DNA in a diploid human cell = 3.3x2 (about 7) pg.

    White blood cells in 1 mL peripheral blood = ˜6-7 x 10e6.

    DNA theoretical yield: 40-50 mcg/mL blood.

    DNA used in a Southern blot: 5-10 mcg.

    DNA used in one PCR reaction: 0.1 mcg.

    Protocols for Nucleic Acid Extraction [Human] - PubMed - DNA - RNA

    Salting out: Miller et al. 1988


    DNA