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Chapter 1 - Purification, General Workflow and Isolation Techniques -…

- - - - Many applications require purified DNA such as
        
        Tissue typing for organ transplant
        
        Detection of pathogens
        
        Human identity testing
        
        Genetic research
    - - Separation from proteins, lipids, RNA, etc...
      - Fragmentation of long DNA
        
        Mechanical Shearing
        
        Endogenous Nucleases (DNases)
        
        How do you stop DNases?
        
        Inactivated by heat or chelating agents
    - - Effectively disrupt cells or tissues using usually detergent
        
        Disrupts Hydrogen Bonds
        
        Ionic detergents, such as SDS
        
        Chaotropic agents such as urea and guanidine
        
        Heat
        
        Disrupts Hydrophobic interactions
        
        Chaotropic agents such as urea and guanidine
        
        Ionic detergents, such as SDS
        
        Disrupts Disulfide bonds
        
        Reducing agents (DTT)
        
        Disrupts Non-polar interactions
        
        Heat
      - Denature proteins/nucleoproteins using proteases/denaturant such as proteinase K
      - Inactivate endogenous nucleases(chelating agents)
      - Purify nucleic acid target away from other nucleic acids and protein
        
        using RNases, proteases, selective matrix and alcohol precipitations
    - - Photometric measurement of DNA concentration
        = UV 260 nm
      - Conc = 50xOD260
  - - - Messenger RNA (mRNA): 1-5%
        
        Serves as a template for protein synthesis
      - Ribosomal RNA (rRNA): >80%
        
        Structural component of ribosomes
      - Transfer RNA (tRNA): 10-15%
        
        Translates mRNA information into the appropriate amino acid
    - - Cells or tissue must be rapidly and efficiently disrupted
      - Inactivate RNases
      - Denature nucleic acid-protein complexes
      - RNA selectively partitioned from DNA and protein
    - - Properties
        
        Naturally occurring enzymes that degrade RNA
        
        Common laboratory contaminant (from bacterial and human sources)
        
        Released from cellular compartments during isolation of RNA from biological samples
        
        Can be difficult to inactivate
      - Protection against RNases
        
        Wear gloves at all times
        
        Use RNase-free tubes and pipet tips
        
        Use dedicated, RNase-free, chemicals
        
        • Pre-treat materials with extended heat (180°C for several hours), wash with DEPC-treated water, NaOH or H2O2
        
        Supplement reactions with RNase inhibitors
        
        Include a chaotropic agent (guanidine) in the procedure as Chaotropic agents such as guanidine inactivate and precipitate RNases and other proteins.
    - - Organic Extraction
        
        How does it work?
        
        Step 1 : Lyse cells Add pheonl:chlorofoam:isoamyl alcohol to the lysed sample
        Step 2 : Centrifuge
        Step 3 : Separation occurs
        
        Organic solvents on the bottom
        
        Aqueous phase on top (contains total RNA)
        
        Cellular debris and genomic DNA will separate the two solutions and act as a 'film'
        
        Step 4 : Remove RNA solution to a tube
        Step 5 : Precipitate RNA and wash with ethanol
        Step 6 : Resuspend RNA in water
        
        At pH levels high than 7.0, RNA and DNA partition into aqueous phase
        
        At pH levels lower than 7.0, DNA is denatured and will move into the organic phase, but the RNA remains in the aqueous phase
        
        Advantages
        
        Scalable - can process small and large samples
        
        Established and proven technology
        
        Inexpensive
        
        Versatile - compatible with a variety of sample types
        
        Disadvantages
        
        Not High-throughput
        
        RNA may contain contamining genomic DNA
        
        Organic solvents
      - Affinity Purification
        
        How does it work?
        
        Step 1 : Lyse cells
        Step 2 : Spin to remove cell debris
        Step 3 : Apply Lysate which contains cellular contaminants and nucleic acids into the column with a glass membrane
        Step 4 : Wash the column with alcohol to remove contaminants so that the nucleic acids stick to the membrane whereas the contaminants are flushed through the glass membrane
        Step 5 : Treat with DNase to remove DNA
        Step 6 : Add water to column; the purified RNA is flushed through the membrane
        
        Advantages
        
        Eliminates need for organic solvents
        
        Compatible with a variety of sample types (tissue, tissue culture cells, white blood cells, plant cells, bacteria, yeast, etc.)
        
        DNase treatment eliminates contaminating genomic DNA
        
        Excellent RNA purity and integrity
      - Alternative nucleic acids purification protocol
        
        CsCl gradient centrifugation
        
        The best quality of RNA obtainable
        
        RNA and DNA isolated simultaneously
        
        Has a 36h centrifugation step
    - - Photometric measurement of RNA concentration = UV 260 nm
      - Conc=40xOD260
- - - - separation of DNA from proteins and cellular debris
    - - Organic extraction
        
        How does this work?
        
        DNA is polar -> not soluble in organic solvents
        
        When phenol is mixed with DNA. DNA partitions to the (upper) aqueous phase, denatured proteins partition to the (lower) organic phase.
        
        Since DNA is polar, This polarity makes it more soluble in the polar aqueous phase.
        
        What do we use?
        
        phenol:chloroform
      - Salting out
        
        What do we use?
        
        1:1 phenol : chloroform (or)
        25:24:1 phenol : chloroform : isoamyl alcohol
        
        How does this work?
        
        Phenol: denatures proteins, precipitates form at interface between aqueous and organic layer
        
        Chloroform: increases density of organic layer
        
        Isoamyl alcohol: prevents foaming
      - Selective DNA binding to a solid support
        
        How does this work?
        
        Separates DNA from crude cell lysates by selective binding to a support material.
        
        What do we use?
        
        Silica
        
        Anion-exchange resin
        
        Advantages
        
        Speed and convenience
        
        No organic solvents
        
        Amenable to automation/miniaturization
        
        Disadvantages
        
        DNA Fragmentation
  - - - Step 3 : Quality parameters } Purity & Integrity
        
        Step 2 : RNA Purification
        
        Step 1 : Break open cell/tissue