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    DNA Isolation

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    Yiğit Arslan
    This document discusses DNA isolation and provides details about DNA structure and isolation methods. It describes DNA as a natural polymer that encodes genetic information and is found in all living organisms. It then explains the basic three steps of DNA isolation as cell lysis, removal of proteins/contaminants, and DNA recovery. Common isolation techniques include chemical or mechanical lysis followed by deproteination and DNA precipitation or binding to silica for purification. The document also notes that DNA concentration can be measured spectrophotometrically by absorbance at 260nm.

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    DNA Isolation

    Uploaded by

    Yiğit Arslan
    126 views20 pages
    This document discusses DNA isolation and provides details about DNA structure and isolation methods. It describes DNA as a natural polymer that encodes genetic information and is found in all living organisms. It then explains the basic three steps of DNA isolation as cell lysis, removal of proteins/contaminants, and DNA recovery. Common isolation techniques include chemical or mechanical lysis followed by deproteination and DNA precipitation or binding to silica for purification. The document also notes that DNA concentration can be measured spectrophotometrically by absorbance at 260nm.

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    This document discusses DNA isolation and provides details about DNA structure and isolation methods. It describes DNA as a natural polymer that encodes genetic information and is found in all living organisms. It then explains the basic three steps of DNA isolation as cell lysis, removal of proteins/contaminants, and DNA recovery. Common isolation techniques include chemical or mechanical lysis followed by deproteination and DNA precipitation or binding to silica for purification. The document also notes that DNA concentration can be measured spectrophotometrically by absorbance at 260nm.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    126 views20 pages

    DNA Isolation

    Uploaded by

    Yiğit Arslan
    This document discusses DNA isolation and provides details about DNA structure and isolation methods. It describes DNA as a natural polymer that encodes genetic information and is found in all living organisms. It then explains the basic three steps of DNA isolation as cell lysis, removal of proteins/contaminants, and DNA recovery. Common isolation techniques include chemical or mechanical lysis followed by deproteination and DNA precipitation or binding to silica for purification. The document also notes that DNA concentration can be measured spectrophotometrically by absorbance at 260nm.

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    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 20

    DNA ISOLATION

    Dilek Mercan
    DNA: THE CODE OF LIFE
    Deoxyribonucleic acid (DNA), is a
    natural polymer
    Encodes the genetic information
    required for the growth,
    development, and reproduction of
    an organism
    Is thought to date back to ~3.5-4.6
    billion years ago
    Found in all living organisms
    except RNA viruses
    In complex eukaryotic cells most of the DNA is located
    in cell nucleus
    In prokaryotes DNA is not seperated from cytoplasm
    by nuclear envelope
    Chloroplast and mitochondria also carry DNA



    X-ray diffraction pattern of DNA by Rosalind
    Franklin. 1952
    Double Helix model proposed by Watson
    and Crick, 1953
    Discovery by Friedrich Miescher in 1860s Isolated
    a P-rich material of unknown function from cell
    nuclei

    1952Rosalind Franklin, X-ray diffraction pattern of
    DNA

    1953J. Watson and F. Crick, double helix model
    DNA STRUCTURE
    DNA is a double-
    stranded helix.
    A macromolecule made
    of monomers called
    nucleotides.
    DNA is tightly coiled around proteins called histones
    BASE PAIRING
    (COMPLEMENTARY RULE)
    G and C forms a base pair with 3 hydrogen bonds
    A and T forms a base pair with 2 hydrogen bonds

    DNA IS NEGATIVELY CHARGED
    PO
    4
    -3
    Each strand has a free
    phosphate group at the 5'
    end and a free hydroxyl
    group at the 3' end.
    A routine procedure to collect DNA for subsequent molecular,
    diagnostic or forensic analysis
    DNA can be isolated from;
    Bacteria, yeast, fungi
    Blood or any other cell (except erythrocytes in mammals) from
    animals
    Plants
    Viruses
    Fossil remainings
    DNA ISOLATION
    DNA ISOLATION STEPS
    3 BASIC STEPS

    1. Lysis (cell lysis)

    2. Removal of proteins and contaminants
    (deproteination)

    3. Recovery of DNA (DNA elution)


    Cell lysis: mechanically (beads or sonication) or chemically
    (detergents like SDS or Triton X-100 or enzymes like
    lysozome)
    Seperation of DNA associated proteins and other
    cellular proteins: protein degrading enzymes like
    proteinase K
    DNA elution: precipitated by mixing with cold ethanol and
    then centrifuging. The DNA is insoluble in the alcohol and
    will come out of solution, and the alcohol serves as a wash
    to remove the salt previously added. Then, resuspention in
    TE buffer or dH2O
    DNA samples can be kept at;
    +4C
    -20C
    -80C

    Whole blood samples treated with an anticoagulant
    can be kept at +4 for 10 days. For long term
    storage -80C
    SILICA BASED METHOD
    Selective adsorption of nucleic acids to a silica-gel
    membrane in the presence of high concentrations of
    chaotropic salts (like guanidinium chloride for
    denaturation).
    Use of optimized buffers in the lysis procedure
    ensures that only DNA is adsorbed while cellular
    proteins, and metabolites remain in solution and are
    subsequently washed away.
    DNA is then eluted (released) from the silica-gel
    membrane using a low-salt buffer.
    Chaotropic salts are added to
    the sample to denature the DNA
    by disrupting its hydrogen
    bonding.
    Under these conditions, the
    DNA will selectively bind to the
    silica resin in the column.
    Low salt solution allows the
    renaturation of the DNA, causing
    it to lose affinity for the silica.
    DNA YIELD
    Yield of genomic DNA depends on the number of
    cells in the sample
    A 200 l sample of whole blood from a healthy
    donor (5x10
    6
    leukocytes/ml) will yield approximately
    6 g of DNA
    LAB ASSIGNMENT
    Isolate genomic DNA from whole blood using
    BioBasic EZ-10 Spin Column Genomic DNA
    Minipreps Kit
    Measure the concentration of isolated DNA usign
    spectrophotometric methods.
    CONCENTRATION OF DNA
    Nucleotides absorb 260 nm UV light
    Measurement of purity;
    A260/A280
    Proteins absorb UV light at 280 nm
    A260/A280 ~ 1.8 means pure DNA
    In the presence of impurities like proteins and
    phenol, value decreases.

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