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    L2.1

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    mic 402

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    L2.1

    Uploaded by

    mohammd arif mugheri
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    mic 402

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    © © All Rights Reserved

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    mic 402

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    0 views30 pages

    L2.1

    Uploaded by

    mohammd arif mugheri
    mic 402

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 30

    Purification of DNA

    What are the Structures of the Cell?


    • DNA can be extracted from either plant, animal, bacteria, virus etc.
    • Mostly Chromosomal/genomic DNA. Sometimes plasmids may be present.
    Isolation of DNA is often the first step before further analysis

    • DNA profiling

    • Cloning/PCR

    • Disease diagnosis

    • DNA sequencing

    • Genetically modified organisms (GMO) –


    • agriculture, pharmaceutical

    • Environmental testing, biodefense


    How Much DNA Do We Need?

    •The RFLP procedure requires a minimum of 50 ng of high molecular


    weight double stranded DNA. This is the equivalent of
    approximately 2 ul of blood.
    •The PCR reactions call for on average 1 ng of DNA (single or double
    stranded).
    •This is the equivalent of 1/20 of 1 ul of blood.
    •Many of the commercially available kits are sensitive below 1 ng of
    DNA (100-250 pg).
    How Much DNA Can We Recover?

    • A Diploid Cell contains approximately 6 pg of DNA

    • Sperm contains approximately 3 pg of DNA

    • The average WBC of an adult is 5 - 10 X 106 cells per ml of blood. Therefore, the
    theoretical recovery of DNA per ul of blood is 30 - 60 ng.
    What are the Most Commonly used DNA Extraction
    Procedures
    • Organic (Phenol-Chloroform) Extraction
    • Non-Organic (Proteinase K and Salting out)
    • Chelex (Ion Exchange Resin) Extraction
    • Silica Based (Silica exchange resin- Qiagen)

    The method utilized may be sample dependent, technique dependent, or


    analyst preference
    1. Cell lysis (chemical,
    mechanical, 2. Purification of DNA
    from cell extracts
    or enzymatic)

    Nucleic Acid
    Extraction

    4. Measurement of
    3. Concentration of
    purity and DNA
    the nucleic acids
    concentration
    1. Preparation of a cell extract:
    To extract DNA from a tissue/cells of interest, the cells have to be separated and the cell
    membranes have to be disrupted.
    The "Extraction buffer" helps in carrying out these processes.
    Chemicals such as EDTA (Ethylene Diamine Tetra Acetate) which removes Mg2+ ions that are
    essential for preserving the overall structure of the cell membrane, and SDS (Sodium Dodecyl
    Sulfate) which aids in disrupting the cell membranes by removing the lipids of the cell
    membranes are included in the extraction buffer.
    Having lysed the cells, the final step in the preparation of a cell extract is removal of insoluble cell
    debris.
    Cell debris and partially digested organelles etc. can be pelleted by centrifugation leaving the cell
    extract as a reasonably clear supernatant.
    EXTRACTION REAGENTS

    Cell Lysis Buffer - Non-ionic detergent, Salt, Buffer, EDTA designed to lyse outer cell
    membrane, but will not break down nuclear membrane.
    EDTA (Ethylenediaminetetraacetic disodium salt) is a chelating agent of divalent cations
    such as Mg2+. Mg2+is a cofactor for DNase nucleases. If the Mg2+is bound up by EDTA,
    nucleases are inactivated.
    TE Buffer - Tris-EDTA Buffer: TE buffer is often used to store DNA and RNA. EDTA in TE
    chelates Mg2+ and other divalent metals ions necessary for most causes of DNA and
    RNA degradation, suppressing these processes. Tris is a buffering agent to keep the
    solution at a defined pH.
    EXTRACTION REAGENTS

    Proteinase K - it is usual to remove most of the protein by digesting with proteolytic


    enzymes such as Pronase or proteinase K, which are active against a broad spectrum of
    native proteins, before extracting with organic solvents.

    Protienase K is approximately 10 fold more active on denatured protein. Proteins can


    be denatured by SDS or by heat.
    •Protease is added to destroy nuclear proteins that
    bind DNA and cytoplasmic enzymes that breakdown
    and destroy DNA.
    Why Add Protease?

    •Protease treatment increases the amount of intact


    DNA that is extracted.
    •The protease solution already contains salt

    •Na+ ions of NaCI bind to the phosphate groups of DNA


    Adding Salt molecules, neutralizing the electric charge of the DNA molecules.

    •The addition of NaCI allows the DNA molecules to come together


    instead of repelling each other, thus making it easier for DNA to
    precipitate out of solution when alcohol is added.
    • Non-Organic DNA Extraction
    • Bacterial cells are cultured in liquid media until they reach a maximum density
    of 2-3x109 cells/ml, and then harvested.
    • The E.coli chromosome is just over 4.5 MB in size, amounting to approximately
    .005 picograms per cell. A typical overnight culture from a single starting
    colony will contain approximately 1-2×109 cells/ml. Theoretically, that means
    that 1 ml of culture should yield about 5 µg of gDNA per 109 bacterial cells.
    Take this into account when calculating how much DNA you need for your
    chosen application.
    • The collected cells are lysed, often done physically/chemically, using reagents
    such as lysozyme, EDTA, and detergents, etc.
    • Cellular components are then removed using organic/non-organic extraction
    or silica-based technologies. The final step involves DNA precipitation to
    obtain pure DNA at a high concentration. This procedure can be applied to a
    wide variety of microbes and other unicellular organisms such as yeast.
    • Following cell lysis (which brings the gDNA into solution), the only thing left to
    do is to purify the sample.
    •1. Lysis: Just Crack Them Open

    •Genomic DNA (gDNA) extraction is the simpler procedure because


    strong lysis is the only step necessary to release gDNA into solution.
    •For yeast, plants, and bacteria, lysis involves enzymatically breaking
    the strong, rigid cell wall before mechanically disrupting the plasma
    membrane.
    •Cell walls are usually digestible with lysozyme, which hydrolyzes cell
    wall peptidoglycans, and the serine protease proteinase K.
    •For certain gram-positive species, lysostaphin will further aid
    enzymatic digestion.
    •You may need to use different enzymes for more exotic species with
    different cell wall compositions.
    •Mechanical cell wall disruption represents a more universal lysis
    method for gDNA extraction.
    •Bead beating is popular, and you can easily do this on a vortex using
    0.1 mm glass beads or 0.15 mm fine garnet beads.
    •Special vortex adapters help with performing multiple extractions at
    the same time with equal efficiency.
    •Bead beating is faster than enzymatic lysis and generally more
    thorough.
    •For tough filamentous fungi (e.g. Aspergillus and Fusarium spp.),
    cellular material is often snap-frozen in liquid nitrogen and milled in a
    pestle and mortar followed by rapid vertexing in solution with an
    appropriate lysis buffer.
    Non-Organic DNA Extraction Procedure
    Cell Lysis Buffer - lyse cell membrane, nuclei are intact, pellet
    nuclei. In Tris-EDTA buffer with SDS or lysozyme.

    Resuspend nuclei in Protein Lysis Buffer containing a high


    concentration of Proteinase K.

    Temperature helps denature proteins, and Proteinase K auto


    digests itself

    To remove proteinaceous material, NaCl is added to a final


    concentration of 2.5 M and incubated on ice.
    DNA purification: phenol/chloroform extraction

    1:1 phenol : chloroform


    or
    25:24:1 phenol : chloroform : isoamyl alcohol

    Phenol: denatures proteins, precipitates form at interface between


    aqueous and organic layer

    Chloroform: increases density of organic layer

    Isoamyl-alcohol: prevents foaming


    Genomic DNA prep: removing proteins and
    RNA

    Need to mix gently! (to avoid shearing breakage of the genomic DNA)
    Add the enzyme RNase to degrade RNA in the aqueous layer
    Organic Extraction
    Pros:
    • yields relatively pure, high molecular weight DNA
    • DNA is double stranded – good for RFLP
    Cons:
    • Time consuming
    • Requires sample to be transferred to multiple tubes –
    • increases risk of contamination
    • Involves use of hazardous (and smelly!) chemicals
    2 ways to concentrate the genomic DNA

    70% final conc.

    “spooling” Ethanol precipitation


    Concentrating DNA Alcohol Precipitation
    •The most widely used method for concentrating DNA is precipitation
    with ethanol.
    •The precipitate of nucleic acid, forms in the presence of moderate
    concentrations of monovalent cations (Salt, such as Na+), is
    recovered by centrifugation and redissolved in an appropriate buffer
    such as TE.
    •The technique is rapid and is quantitative even with nanogram
    amounts of DNA.
    •DNA in ethanol precipitates better in the presence of salt.
    •NaAc (or KAc) is well soluble in ethanol, as opposed to for example
    NaCl, thats why it is used for precipitation.
    •An ethanol precipitation with sodium acetate is a concentration/
    wash step, it is not a purification step.
    •Why is chilled ethanol used for DNA extraction?

    •Using ice-cold ethanol and ice-cold water increases the yield of DNA.

    • Low temperatures protect the DNA by slowing down the activity of


    enzymes that could break it apart.

    •DNases in the cytoplasm would destroy the DNA of viruses entering


    the cell. Cold ethanol helps the DNA to precipitate more quickly.
    Concentrating DNA Alcohol Precipitation

    •Solutes that may be trapped in the precipitate may be removed by


    washing the DNA pellet with a solution of 70% ethanol.
    •
    •To make certain that no DNA is lost during washing, add 70% ethanol
    until the tube is 2/3 full. Vortex briefly, and recentrifuged.

    • After the 70% ethanol wash, the pellet does not adhere tightly to the
    wall of the tube, so great care must be taken when removing the
    supernatant.
    Concentrating DNA Alcohol Precipitation

    •Very short DNA molecules (<200 bp) are precipitated inefficiently by


    ethanol.

    •The optimum pelleting conditions depend on the DNA concentration.

    •Relatively vigorous microcentrifuge steps such as 15 minutes at or


    below room temperature at 12,000 rpm are designed to minimize the
    loss of DNA from samples with yields in the range of a few
    micrograms or less.
    What are the essential targets of a DNA extraction Procedure?

    1. Maximize DNA recovery


    2. Remove inhibitors
    3. Remove or inhibit nucleases
    4. Maximize the quality of DNA

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