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    Isolation & Characterization of Dna: Objectives

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    Krizzi Dizon Garcia
    1. Key steps to isolate DNA from samples include homogenization to break open cells, deproteinization to remove proteins, and precipitation to separate DNA from solution using ethanol. 2. DNA concentration and purity can be determined by measuring absorbance at 260nm and 280nm, with a ratio of 1.8 being pure DNA. 3. Chemical tests on DNA hydrolysates can identify components like deoxyribose using diphenylamine, phosphates using ammonium molybdate, purines using murexide, and pyrimidines using Wheeler-Johnson test.

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    Isolation & Characterization of Dna: Objectives

    Uploaded by

    Krizzi Dizon Garcia
    146 views4 pages
    1. Key steps to isolate DNA from samples include homogenization to break open cells, deproteinization to remove proteins, and precipitation to separate DNA from solution using ethanol. 2. DNA concentration and purity can be determined by measuring absorbance at 260nm and 280nm, with a ratio of 1.8 being pure DNA. 3. Chemical tests on DNA hydrolysates can identify components like deoxyribose using diphenylamine, phosphates using ammonium molybdate, purines using murexide, and pyrimidines using Wheeler-Johnson test.

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    Dna

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    1. Key steps to isolate DNA from samples include homogenization to break open cells, deproteinization to remove proteins, and precipitation to separate DNA from solution using ethanol. 2. DNA concentration and purity can be determined by measuring absorbance at 260nm and 280nm, with a ratio of 1.8 being pure DNA. 3. Chemical tests on DNA hydrolysates can identify components like deoxyribose using diphenylamine, phosphates using ammonium molybdate, purines using murexide, and pyrimidines using Wheeler-Johnson test.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    146 views4 pages

    Isolation & Characterization of Dna: Objectives

    Uploaded by

    Krizzi Dizon Garcia
    1. Key steps to isolate DNA from samples include homogenization to break open cells, deproteinization to remove proteins, and precipitation to separate DNA from solution using ethanol. 2. DNA concentration and purity can be determined by measuring absorbance at 260nm and 280nm, with a ratio of 1.8 being pure DNA. 3. Chemical tests on DNA hydrolysates can identify components like deoxyribose using diphenylamine, phosphates using ammonium molybdate, purines using murexide, and pyrimidines using Wheeler-Johnson test.

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    of 4

    KEY STEPS:

    Isolation & - Homogenization (unifying the components


    characterization of your solution; one phase) / Cell lysis
    - Deproteinization
    of dna - Precipitation (separation of DNA from
    solution)
    objectives
    • Homogenizing solution:
    1. Distinguish different components of DNA - Sodium dodecyl sulfate: detergent used to
    based on qualitative tests lyse cell wall, cell membrane, & nuclear
    2. Evaluate the quality and concentration of membrane
    DNA based on absorbance readings - NaCl: stabilizes the negatively charged
    3. Propose a method to isolate DNA from DNA (phosphate group)
    biological samples *when clumped together the surface area
    of the molecule; hence, precipitation
    - Sodium Citrate: buffer (maintain the
    A. Isolation of dna from onion negative charge of DNA)
    - EDTA: chelates metal cofactors of
    DNAses/nucleases (preserve the integrity
    - Onions are commonly used because it of the DNA molecule)
    contains a lot of DNA
    • Blending helps in homogenization (for
    uniformity)
    • Deproteinization – a protease can be added to
    the homogenized solution (to remove proteins;
    increases purity of DNA isolate) ex: papain
    (hydrolyze nucleases)
    • Precipitation – separation of DNA
    -95% cold ethanol (DNA is insoluble at 65%
    EtOH and above)
    *DNA is highly concentrated in the nucleus region *water has a higher dielectric constant
    compared to EtOH; when high, the interaction of
    *The cell wall and cell membrane must be
    the (-) charge of the PO4 & (+) charge of Na
    disrupted to access the DNA
    atoms will be low
    *increase the concentration of EtOH for a
    greater interaction between PO4 & Na
    *EtOH reacts with water and minimizing the
    capability of water to solvate the DNA molecule;
    hence, the DNA will only interact with each other
    for precipitation
    DNA Extraction Kits
    - Follows the same principle, but the
    protocol might vary depending on the
    sample

    - DNA purity can be assessed by taking the


    ratio of absorbances at 260 and 280 nm
    (A260/A280)

    B. determination of dna > A260 = nitrogenous bases


    A280 = aromatic amino acids
    concentration & purity
    >

    DNA Nomograph
    How to use:
    1. Plot the absorbances at A260 and A280 at
    its respective lines (3rd and 2nd lines in the
    - Due to absorbance of the aromatic rings nomograph)
    of the nitrogenous bases 2. Connect both points and extend the line
    - Strongest at 260 nm towards the outer lines to determine the
    A260 ∝ [DNA] protein and nucleic acid concentration
    *The aromatic rings nitrogenous bases
    have the maximum absorbance at 260 nm
    - The concentration of a pure DNA sample
    can be estimated by:

    Conc (𝜇g/mL) = (A260 reading) x dilution factor x 50 𝜇g/mL

    *50 𝜇g/mL is a constant which is the molar


    absorptivity of DNA
    Positive result Blue-dark blue solution
    C. acid hydrolysis of dna

    - Hydrogen bonds between complementary


    bases were cleaved
    *DNA would melt; strands would separate
    - Depurination would occur in mild acidic
    conditions
    *purine nitrogenous base would be cleaved
    from the nucleotide/nucleoside
    - A nucleotide undergoes complete hydrolysis *Intensity of blue color is directly proportional to
    when subjected to acid hydrolysis the concentration of DNA (deoxyribose)
    > Inorganic phosphate
    > 2-deoxyribose
    b. Test for phosphate (Ammonium
    > Nitrogenous bases
    molybdate test)
    -Differentiates nucleotide from nucleoside
    Reagent Conc HNO3,
    (NH4)2MoO4, (heat)
    Principle Complexation
    Detects Inorganic phosphate
    Positive result Yellow ppt
    d. chemical characterization
    of dna

    DNA HYDROLYSATE + Test reagent/procedure

    a. Test for Deoxyribose


    (Diphenylamine/Dische Reaction)
    Reagent Diphenylamine (acidic)
    Principle Condensation
    Detects Deoxyribose
    c. Test for purines (Murexide test)
    Reagent Conc HNO3, KOH, or
    NH4OH
    Principle Condensation
    Detects Purines
    Positive result Yellow to reddish ppt
    *any purine

    d. Test for pyrimidines (Wheeler-Johnson


    test)
    Reagent Ba(OH)2, bromine
    water
    Principle Bromination, then
    hydroxylation
    Detects Pyrimidines (except
    thymine)
    Positive result Yellow then purple
    solution
    *followed by rearrangement

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