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DNA Isolation


In this laboratory you will learn how to isolate the hereditary molecule and blue-print
molecule of life called DNA from different biological sources. You will use two different
methods to isolate DNA from plant material and bacteria. You will further learn how to
visualize DNA and quantify the amount of isolated DNA using DNA-
specific dyes and UV spectrophotometry

Laboratory Objectives

After completion of this lab you should:

1.  have a deep understanding of the composition, structure and function of DNA

2. have good knowledge and gained skills in methods to extract and
isolate DNA from biological material, including bacteria, plant and animal cells

3. have a good working knowledge with different DNA staining dyes and methods

4. be familiar with methods and techniques to separate and visualize DNA

5. know how to use a spectrophotometer to determine the amount of DNA in a given
sample (= DNA quantitation)

Introduction:

•    Deoxyribonucleic acid or simply DNA, is the molecular foundation of the
     sustainability of life on planet Earth

•   The nucleic acid molecule DNA is absolutely crucial for growth, reproduction,
     adaptation, and survival of all life forms, whether a simple bacterium, amoeba,
     slime molds, fungi, plants or animals.

•   DNA is the genetic material of all life forms on planet Earth; it bears the code for
all biological structures and it absolutely crucial for the successful production of all
cellular components, i.e. proteins, enzymes and molecules derived thereof
-
exceptions are certain viruses, so-called RNA or Retro-viruses, which relay on the
  nucleic acid RNA as their genetic material
- but biologists don’t consider viruses as true life forms

•   DNA is a molecule which is made up from two polynucleotide strands which are wrapped
    around each other to form a so-called double helix
structure (see Graphic 1 below);
    - each poly-nucleotide strand is made up from from 4 structurally different so-called nitrogenous
      bases, which are
adenine (A), thymine (T), cytosine (C) and guanine (G)
    - the follow-up of these four nucleotides, the so-called nucleotide sequence, of the
      polynucleotide strand is the chemical foundation of the genetic code of life
    - the individual nucleotides in each of the two polynucleotide strands are covalently linked
      together
via formation of a phosphodiester linkage between the 3'-hydroxyl group of the
      deoxyribose sugar of one nucleotide and the 5'-phosphate group of the second nucleotide
    - repeated linkage between consecutive nucleotides forms the sugar-phosphate backbone
      
of the polynucleotide strand

Graphic 1: Components and 3-dimensional structure of the DNA molecule

 

•  The DNA molecule made a furious career in the past 140 years since its humble discovery by
Friedrich Miescher in the 1860s; he isolated a phosphate-rich material of (by that time)
unknown function from cell nuclei of collected pus material while working at the University
of Tuebingen in Germany

•   It took another 80 years before in 1943, Oswald Avery, Colin McLeod and Maclyn McCarty
    (working at the Rockefeller University in New York), were finally able to demonstrate that
     DNA and not proteins or carbohydrates carry the genetic code of life

•   Avery and his team discovered that DNA was the transforming agent which changed (=
    transformed) a non-pathogenic bacterial strain of the disease-causing bacterium
    Streptococcus pneumoniae
into a the pneumonia causing strain; DNA and not other cell
    components therefore must have contained the information for the illness-causing factor

•   The announcement of the three-dimensional structure of the DNA molecule as a double-helix
     by James Watson & Francis Crick with the help of other pioneering scientists in Cambridge,
     England in 1953 did not only change our basic understanding of the storage, flow and
     processing of information in living organisms but also the scope and direction of future
     investigations in modern biology

•   Since its first discovery and isolation by F. Miescher more than 100 years ago, scientists
    developed more and more sophisticated methods to isolate, manipulate and visualize nucleic
    acids, such as DNA and RNA

•   Avery in the 1940s succeeded to identify DNA as the blueprint molecule of heredity because
    of his
laboratories’ skills in extracting, purifying and identifying biological molecules from
    cells, including DNA, proteins and carbohydrates - skills which are indispensable for anyone
    working in a modern molecular biology laboratory today!

•   Extraction and purification of cellular molecules today involves numerous chemical and physical
     techniques
, including:

            1. Mortars & Blenders
                - they create mechanical shear stress to rupture the rigid cell walls of plant
                  cells and bacteria and tear the fibers of connective tissue

            2. Detergents
                - detergents such as SDS, sodium desoxycholate or Tween-20, destabilize and
                  destroy the fragile phospholipid-made cell membranes to
release the cell contents

            3. Enzymes
                - addition of protein-degrading enzymes (= proteases), RNA-degrading enzymes
                  (RNAses) to cells and cell lysates help to degrade and to get rid off of certain,
                  unwanted molecules
                    - important proteases used are: Proteinase K or Papain

            4. Filters, Sieves & Dialysis membranes
                - help to mechanically separate molecules and cell components according to size

            5. Centrifuge
                - is a lab equipment that is able to create high gravitational (G)-forces with the
                  help of a spinning rotor which helps to mechanically separate molecules
                  according to their molecular weight (MW) and their size

            6. Chemicals
                - are usually organic solvents such as phenol and chloroform, which are added to
                  crude cell extracts (lysates) to extract proteins
                - ethanol, acetone and salts are commonly used to precipitate DNA and proteins

•    Good knowledge and skills in extraction and purification methods was and still is at the
     center stage of great discoveries (such as the one of Avery and colleagues) in modern
     Biology

•    Successful DNA isolation requires sequential steps which are:

1.      Destruction of the cells by gentle means by a process called cell lysis
   
- this is achieved by using mechanical (use of mortar & pistil) or sonic waves
      (use of a sonicator device) to disrupt the cell integrity and to release the cell
      contents into the
lysis buffer
    - it can also be achieved by using detergents, such as SDS or Triton X-100, which
       disintegrate the lipid-made cell membrane

2.      Separation of other cell constituents, most importantly proteins and lipids
    (= Deproteination)
      - this is routinely
achieved by adding protein-degrading enzymes, such as
        papain (see first lab part) or proteinase K (see second lab part)
     - it is further achieved by the adding organic solvents, such as phenol, chloroform
       and isoamyl alcohol to the cell lysate

3.      Precipitation, trapping and recovery of the DNA molecule (DNA elution)
     -
“DNA clumping” or precipitation is achieved by the addition of cold ethanol
      - DNA is trapped (collected) with the help of positively charged silica material,
        such as diatomaceous earth

4.      DNA identification and estimation of the recovered amount of DNA
    (= DNA Quantitation) usually with the help of dyes and/or a spectrophotometer

•   In this exciting lab you have the opportunity to isolate (extract), visualize, identify and
 quantitate DNA from different organisms 

•   The lab concepts include the extraction of plant DNA from an onion plant, and of genomic
     DNA from bacterial or buccal cells, its digestion with restriction endonucleases and visualization
     with the help of DNA-attaching (- intercalating) dyes.

•   In the first part of this Bio210A lab section you will learn some of the “classical”
    methods and lab procedures used by Miescher, Avery and many other biologists in the
    past to successfully extract and isolate DNA from biological cells
    -
rather than extracting DNA from less appealing pus or dangerous bacteria (as
      Miescher and Avery did), you will extract DNA from easily available and harmless
      onion cells

•   You will further perform a simple qualitative test using a DNA-binding dye called
     diphenylamine to proof that the material you isolated from the onion cells is actually
     DNA

•   In the first part of this lab you will further determine the amount of DNA isolated from
     the onion cells with the help of a spectrophotometer ; you will further use
this
     i
mportant laboratory equipment to perform simple  quantitative tests with your
     extracted onion DNA (see
Procedure 1 below for more details about the individual steps)

•   In the second part of this lab you will finally learn how to isolate chromosomal DNA
     from bacterial cells using the Qiagen silica spin column method, a modern and rapid
     DNA isolation method, which relays on the binding of the negatively charged DNA
     molecule to a charged, porous silica gel material
(see Procedure 2 below for more
     details about the individual steps of this second DNA isolation method)

•   After completion of the second DNA isolation method, you will (1) either digest the
     isolated bacterial genomic DNA with DNA-cutting enzymes, so-called restrictions
      endonucleases
and then separate the DNA fragments with the help of an important
     molecular biology technique called agarose gel electrophoresis (
see separate
     “Electrophoresis” lab manual) and in a second lab part use the isolated genomic
     bacterial DNA for later amplification of selected genes with the help of the
    
PCR method (see separate "PCR Lab" section)
     - your instructor will tell you what you will be doing with the isolated bacterial DNA

•   Upon successful completion of this lab, you will be one of the relatively small number
    of humans who have had the opportunity to actually “see” DNA and to intimately
    witness their own DNA as the molecular foundation of their biological individuality

•   Beyond the scope of today’s lab you will further understand that DNA isolation is one
     of the fundamental techniques of modern molecular biology and crucial for a series of
     other important DNA processing and analyzing techniques

•   Today, the most important methods which relay on isolated DNA molecules and which
     are routinely used by molecular biologists are:

  1. DNA manipulation, e.g. cutting, fusing together (= ligation), with the help of special
    enzymes, called restriction endonucleases and ligases
  2. DNA sequencing, i.e. reading of the DNA letter code, with the help of a technique
    called Terminal Dideoxy-Sequencing
  3. DNA multiplication (= amplification) with the help of a sophisticated bio-technique,
    called Polymerase Chain Reaction or short: PCR
  4. DNA separation with the help of the powerful analytical method called
    horizontal
    Agarose gel electrophoresis
  5. DNA visualization with the help of colored or fluorescent dyes, such as
    diphenylamine or ethidium bromide; the latter in combination with UV light
    produced by a device called an UV transilluminator

•   Today DNA can be extracted and isolated from any type of cell and biological material,
     but different types of cells (e.g. bacteria, plant cells, animal cells) require slightly
     different extraction techniques depending on their size, cell membrane composition
     and cell surrounding structures, e.g.  cell walls or other protective layers

The very simple procedure you will learn in the first part of this "DNA Isolation"
lab enables the extraction of DNA from most types of plant cells. Since plant cells
are surrounded by a sturdy cell wall made of cellulose and other polysaccharides,
plant cell destruction requires strong mechanical forces achieved by either mincing,
blending
or grinding of the plant material in the presence of a mild detergent

•   In the first lab exercise you will therefore learn how to extract DNA from onion plant cells
   
 using a simple and straight forward method as described in the "Procedure 1" below
    - to access the file of Procedure 1 simply click on the Interactive Button below

•   After successful completion of the DNA extraction from the onion material, you
    will receive a whitish, viscous-slimy material; to determine whether the white, slimy
    material you isolated and spooled onto the glass rod is indeed DNA you will have to
    run a simple DNA identification test.

•   There principally two basic types of chemical tests used in science to identify the
     presence of a certain molecule:

    1. Qualitative Tests

    - simply determine IF a substance/molecule X is present in a mix or
  solution
- they are based on specific reactions of the molecule X with a dye or
  chemical which leads to color changes or emission of light

    1. Quantitative Tests

    - accurately determine HOW MUCH of a substance/molecule X is present
  in a mix or given solution
- usually requires the knowledge of the molecule X and its physico-chemical
  properties, e.g. extinction coefficient, molecular weight
- requires usually the establishment of a so-called standard curve which
  helps to figure out the exact amount of the molecule X in the sample
  (see Section 3 of this lab manual for further details)

•    In the last part of exercise 1 (see Procedure 1 file below) you will perform a qualitative
      test on the previously spooled and collected material in your 10ml test tube to determine
      if this material is indeed DNA.  You will use the chemical and DNA indicator dye
       diphenylamine
(for details regarding structure and function of this dye see Graphic 2 below)
       - Diphenylamine (DPA) is a colorless to gray chemical, that is widely used in
         rubber processing chemicals, pharmaceuticals and as an additive for petroleum
         and plastic products.

Lab Biohazard Warning:
DPA is a possible mutagen and a possible teratogen. It is harmful in contact
with skin and if swallowed or inhaled. DPA is an irritant.
Please make sure that you wear vinyl or latex gloves, goggles and a lab coat at
any time while working with this substance!

•   In the second part of this "DNA Isolation" lab you will be introduced to one of the
     most popular and important DNA isolation method which is routinely used in many
     Molecular Biology, Forensics and Genetics labs worldwide
     (see "Procedure 2" file below.

•   The so-called silica spin column method is simple, fast and is based on the
     strong interaction of the negatively charged DNA molecule with the positive
     net charges of highly porous silicon dioxide (Si-O2) filter material (silica gel
     membrane) and its reversible binding to the silica material
     (for an overview see Graphic 2 below)

    Graphic 2: Principle and tools of the Silica Spin Column Technology
                                (How do the QIAamp silica spin columns work?)

 

    Principle: Silica spin column DNA isolation

-   The discovery of the strong binding of DNA to silica materials, such as
diatomaceous earth, was a great leap forward in the efforts of scientists to
find better (and faster) ways to isolate DNA from biological samples

-   Modern silica-based spin columns contain solid, but porous, silica membranes
made up of the SiO4-rich exoskeletons of dead/fossilized biological organisms
called diatoms (therefore: diatomaceous earth!)

-   The negative net charges of the phosphate groups of the DNA and nucleic
acids are absorbed to positive charges of the silica (SiO4) gel membranes of
the spin columns in the presence of high concentrations of chaotropic salts and
low pH values (see Graphic 2 above)

-   The silica binding of DNA is reversible and therefore DNA can be freed (eluted)
from the silica material by simply changing the pH and the salt concentration

-   DNA binding to the silica matrix is strong at alkaline pH and high salt concentrations
and low at acidic pH values and low salt
 

•   You will apply this silica spin column technology and isolate genomic DNA from previously
     cultivated bacterial cells using so-called silica spin columns (see Graphic 2 above)
     which come with a commercial DNA isolation kit

•    Even though there are many commercial kits, based on the same silica membrane
     technology, available these days, you will use the so-called QIAamp DNA Mini Kit
     which is manufactured and commercialized by the Biotech company Qiagen during this lab

•    This kit enables the isolation of very pure total DNA (= genomic, viral, mitochondrial)
    
 from a variety of different cells and cell types, including viruses, bacteria, whole blood,
     plasma, lymphocytes, cultured cells, body tissues and forensic specimen
       - the kit works with tiny amounts of sample material
       - between 5 – 100 μg of DNA can be isolated from up to 50 mg of tissue
         (muscle, liver, heart, brain), swabs (buccal, nasal, pharyngeal) or up to
         200 µl of sampled body fluids with this kit
       - the hands-on preparation time (after cell lysis) is about 20 minutes

•   The DNA isolated with the QIAamp DNA Mini Kit is very pure because the DNA
     material once bound to the silica column membrane in the presence of chaotropic
      salts
can be thoroughly washed by applying washing buffers to get rid of contaminating
     and unwanted cell components, such as proteins, lipids, etc.

•    The purified DNA is finally released (or eluted) from the silica matrix by applying a
     low salt so-called elution buffer

•    Enough of reading about this exciting DNA isolation technology. Why don’t we get
     started and see how we can apply our knowledge about this DNA isolation method
     works in your hands

After you printed out the files for the Lab Procedure 1 (= onion cell DNA isolation)
and Lab Procedure 2 (= Bacterial DNA Isolation) (see section below) and before
you get ready to conduct your experiments, make sure that you have all the
necessary tools and materials ready for use on your bench by check marking the
Equipment & Materials list which is part of your Procedure document.
Have fun and good luck with the experiments!

The files for the upper lab section's Lab Procedures & Summary Questions can
be retrieved by clicking on the "Interactive Buttons" below

Procedure 1        Procedure 2        Summary Q

Important: Make sure that you bring a printed out version of both "Lab Procedure"
documents and of the
"Lab Summary Questions" sheet with you to
the scheduled lab meeting!
(Both completed sheets are mandatory part of the weekly lab report!)