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4.4 Biotechnological Tools and Techniques

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Title: 4.4 Biotechnological Tools and Techniques


1
4.4 Biotechnological Tools and Techniques
  • Recombinant DNA Gel electrophoresis

2
Recombinant DNA
  • Cutting DNA fragments from different sources and
    recombining them together
  • Cutting DNA fragments from different sources and
    recombining them together
  • 4.4.7 State that, when genes are transferred
    between species, the amino acid sequence of
    polypeptides translated from them is unchanged
    because the genetic code is universal. Obj. 1
  • Purpose
  • To investigate genetic disorders
  • Production of drugs (ie. insulin)

3
What complications do you foresee?
  • Consider
  • The size of DNA
  • Where to cut?
  • How to put back together?
  • 4.4.8 Outline a basic technique used for gene
    transfer involving plasmids a host cell
    (bacterium, yeast or other cell), restriction
    enzymes (endonucleases) and DNA ligase. Obj. 2

4
1. Restriction Endonucleases
  • Also known as restriction enzymes
  • Essentially are molecular scissors
  • Recognize a specific DNA sequence and cuts the
    strands at a particular position or recognition
    site
  • Isolated and purified only from bacteria
  • Name reflects which bacteria the enzyme
    originates
  • ie. EcoRI ? Escherichia coli, strain R, 1st r.e.
    isolated
  • HindII ? Haemophilus influenzae, strain Rd, 2nd
    r.e.

5
1. Restriction Endonucleases Recognition site
Bacteria Restriction Enzyme Recognition Site
Escherichia coli EcoRI 5-GAATTC-3 3-CTTAAG-5
Haemophilus parainfluenzae HindIII 5-AAGCTT-33-TTCGAA-5
Arthrobacter luteus AluI 5-AGCT-33-TCGA-5
  • Each restriction endonuclease recognizes its
    own specific recognition site (specific DNA
    sequence)
  • Usually 4-8 base pairs long, characterized by a
    complementary palindromic sequence

6
1. Restriction Endonucleases Function
  • Scans DNA and binds to its specific recognition
    sequence
  • Disrupts the phosphodiester bonds between
    particular nucleotides through a hydrolysis
    reaction
  • Hydrogen bonds of the complementary base pairs in
    between the cuts are disrupted
  • Result 2 DNA fragments

http//www.scq.ubc.ca/?p249
7
1. Restriction Endonucleases DNA Fragment Ends
  • Different DNA fragment ends are produced after
    digestion by different restriction enzymes
  • Sticky ends DNA fragment ends with short
    single-stranded overhangs (ie. EcoRI, HindIII)
  • Blunt ends DNA fragment ends are fully base
    paired (ie. AluI)

Bacteria Restriction enzyme Recognition site After digestion by restriction enzyme
Escherichia coli EcoRI 5-GAATTC-3 3-CTTAAG-5 5-G AATTC-3 3-CTTAA G-5
Haemophilus parainfluenzae HindIII 5-AAGCTT-33-TTCGAA-5 5-A AGCTT-33-TTCGA A-5
Arthrobacter luteus AluI 5-AGCT-33-TCGA-5 5-AG CT-33-TC GA-5
8
1. Restriction Endonucleases DNA Fragment Ends
(continued)
  • http//highered.mcgraw-hill.com/olc/dl/120078/bio3
    7.swf

Restriction site
Animation
Palindrome
Fragment 2
Fragment 1
http//www.bio-rad.com/LifeScience/docs/Official_C
rime_Scene_PowerPoint_Spring_2005_rev_B.ppt
9
How do we control the snips?
  • Consider
  • What about the organisms own DNA?
  • Frequency of recognition sequences within the DNA
    sequence

10
1. Restriction Endonucleases Length of
recognition sites
  • http//highered.mcgraw-hill.com/olcweb/cgi/pluginp
    op.cgi?itswf535535/sites/dl/free/0072437316
    /120078/bio38.swfEarly Genetic Engineering
    Experiment
  • Longer recognition sites result in lower
    frequency of cuts
  • EcoRI ? 5-GAATTC-3 ¼ ¼ ¼ ¼ ¼ ¼
    1/4096
  • AluI ? 5-AGCT-3 ¼ ¼ ¼ ¼ 1/256
  • Higher frequency of cuts may cut gene into
    several fragments
  • Lower frequency of cuts may produce large
    fragments than desired

11
1. Restriction Endonucleases Methylases
  • Enzymes that add a methyl group to a nucleotide
    in a recognition site to prevent restriction
    endonuclease from cutting DNA
  • Distinguishing between foreign (viral) DNA and
    bacterias own DNA

12
1. Restriction Endonucleases DNA Ligase
  • Enzyme that rejoins cut strands of DNA together
    by reforming a phosphodiester bond
  • DNA ligase joins sticky ends
  • T4 DNA ligase (from T4 bacteriophage) joins blunt
    ends

13
How do we sort out the DNA
  • DNA is chopped into many pieces
  • How to differentiate one piece from other

14
2. Gel Electrophoresis
  • Technique used to separate charged molecules
    based on their size
  • 4.4.3 State that gel electrophoresis of DNA is
    used in DNA profiling Obj. 1
  • Acts like a molecular sieve

http//www.biotech.iastate.edu/ppt_presentations/h
tml/Fingerprinting/StudentInstruction-gel/images/i
mage08.jpg
http//www.solve.csiro.au/1105/img/sieve-bloke.jpg
15
DNA Profiling
  • 4.4.4 Describe the application of DNA profiling
    to determine paternity and also in forensic
    investigations. Obj. 2
  • A process of using DNA fragments to identify a
    person, or other organism. The DNA fragments
    have distinct bands separated by spaces and these
    band patterns are so distinct that they can be
    used like fingerprints.

16
2. Gel Electrophoresis DNA Preparation
  • Restriction enzymes digest DNA into smaller
    fragments of different lengths
  • Different DNA samples are loaded into wells of
    the gel (agarose or polyacrylamide)

http//www.oceanexplorer.noaa.gov/explorations/03b
io/background/molecular/media/gel_plate_600.jpg
17
2. Gel Electrophoresis Attraction Migration
  • Negatively charged electrode at the end where
    wells are located
  • Positively charged electrode at opposite end
  • Negatively charged DNA migrate towards positive
    end due to attraction

18
2. Gel Electrophoresis Rate of Migration
  • 4.4.2 State that, in gel electrophoresis,
    fragments of DNA move in an electric field and
    are separated according to their size. Obj. 1
  • Shorter/smaller DNA fragments migrate through gel
    faster since they can move through the pores in
    the gel more easily
  • Longer/larger DNA fragments migrate through gel
    slower
  • Rate of migration 1/log(size)
  • Different DNA fragment lengths are separated

A B C D E
A kilobase DNA ladder B uncut plasmid DNA C
single digestion of the plasmid with EcoRI D
single digestion with XhoI E double digestion -
both EcoRI and XhoI.
http//www.answers.com/topic/agarosegel-jpg
19
2. Gel Electrophoresis Visualizing DNA Fragments
  • Ethidium bromide is a fluorescent dye that makes
    DNA fragments visible by staining the gel
  • DNA fragments can then be isolated and purified

http//www.answers.com/topic/agarosegel-jpg
20
(No Transcript)
21
Paternity Testing
22
4.4.5 Analyse DNA profiles to draw conclusions
about paternity or forensic investigations. Obj.
3
23
Animations
  • http//www.sumanasinc.com/webcontent/animations/co
    ntent/gelelectrophoresis.html
  • http//learn.genetics.utah.edu/content/labs/gel/
  • http//www.dnalc.org/resources/animations/gelelect
    rophoresis.html

24
2. Gel Electrophoresis Proteins too!
  • Gel electrophoresis can also be used to separate
    proteins, usually using polyacrylamide gels

http//www.biotechlearn.org.nz/var/biotech/storage
/images/multimedia/images/protein_electrophoresis/
48251-4-eng-GB/protein_electrophoresis_medium.jpg
http//www.bio-link.org/vlab/Graphics/Tools/Protei
nGel2.jpg
25
3. Plasmids
  • Small, circular double-stranded DNA that can
    enter and exit bacterial cells
  • Lack a protein coat
  • Independent of bacterial chromosome
  • 1000-200,000 base pairs

26
3. Plasmids Endosymbiosis
  • Use host bacterial enzymes and ribosomes to
    replicate and express plasmid DNA
  • Carry genes that express proteins to protect
    bacteria against antibiotics and heavy metals

27
3. Plasmids
  • Foreign genes (ie. insulin) can be inserted into
    plasmids, so bacteria can express gene and make
    its respective protein
  • Higher copy number of plasmids (number of
    individual plasmids) in bacteria
  • results in larger number of gene copies, thus
    more of its respective protein is synthesized

28
3. Plasmids
  • Restriction endonucleases splice foreign genes
    into plasmids
  • DNA ligase reforms phosphodiester bond between
    the fragments, resulting in recombinant DNA
  • http//www.learner.org/courses/biology/archive/ani
    mations/hires/a_gmo1_h.html

http//www.accessexcellence.org/RC/VL/GG/inserting
.html
29
4. Transformation
  • Introduction of foreign DNA (usually a plasmid)
    into a bacterium
  • Plasmids can be used as a vector (vehicle that
    DNA can be introduced to host cells) to carry a
    specific gene into a host cell

http//www.bio.davidson.edu/Courses/Molbio/MolStud
ents/spring2003/Siegenthaler/fig2.gif
30
4. Transformation Competence
  • Competent cell - Bacterium that readily takes up
    foreign DNA (ie. able to undergo transformation)
  • Most cells are not naturally competent, but can
    be chemically induced to become competent
  • Calcium ion in calcium chloride stabilizes
    negatively charged phosphates on bacterial
    membrane

31
4. Transformation Competence
32
4.4.9 State two examples of the current uses of
genetically modified crops or animals. Obj. 1
  • The transfer of a gene for factor IX which is a
    blood clotting factor, from humans to sheep so
    that this factor is produced in the sheeps milk.
  • The transfer of a gene that gives resistance to
    the herbicide glyphosate from bacterium to crops
    so that the crop plants can be sprayed with the
    herbicide and not be affected by it. 

33
  • Issues Surrounding Genetically Modified (GM)
    Products
  • by Subhuti Dharmananda, Ph.D., Director,
    Institute for Traditional Medicine, Portland,
    Oregon

34
Meet the Super Cow
  • http//www.youtube.com/watch?vNmkj5gq1cQU

35
4.4.10 Discuss the potential benefits and
possible harmful effects of one example of
genetic modification. Obj. 3
  • Benefits
  • Crops
  • Enhanced taste and quality, Reduced maturation
    time, Increased nutrients, yields, and stress
    tolerance, Improved resistance to disease, pests,
    and herbicides, New products and growing
    techniques
  • Animals
  • Better yields of meat, eggs, and milk
  • Improved animal health and diagnostic methods
  • Increased resistance, productivity, hardiness,
    and feed efficiency
  • Environment
  • "Friendly" bioherbicides and bioinsecticides
  • Conservation of soil, water, and energy
  • Bioprocessing for forestry products
  • Better natural waste management
  • More efficient processing
  • Society
  • Increased food security for growing populations

36
Controversies
  • Safety
  • Potential human health impact allergens,
    transfer of antibiotic resistance, unknown
    effects.
  • Potential environmental impact unintended
    transfer of transgenes through cross-pollination,
    unknown effects on other organisms (e.g., soil
    microbes), and loss of flora and fauna
    biodiversity
  • Access and Intellectual Property
  • Domination of world food production by a few
    companies
  • Increasing dependence on industrialized nations
    by developing countries
  • Biopiracy-foreign exploitation of natural
    resources
  • Ethics
  • Violation of natural organisms' intrinsic values
  • Tampering with nature by mixing genes among
    species
  • Objections to consuming animal genes in plants
    and vice versa
  • Stress for animals
  • Labeling
  • Not mandatory in some countries (e.g., United
    States)
  • Mixing GM crops with non-GM confounds labeling
    attempts
  • Society
  • New advances may be skewed to interests of rich
    countries
  • Dharmananda, S. Issues Surrounding Genetically
    Modified (GM) Products. Online
    http//www.google.ca/imgres?imgurlhttp//www.itmo
    nline.org/image/gmo1b.jpgimgrefurlhttp//www.itm
    online.org/arts/gmo.htmusg__y5yFCjq561X8N0vAy5g8
    nid85NMh453w400sz32hlenstart5zoom1tb
    nidKqfgq2YL9RdYEMtbnh127tbnw112eik9TfTpasG
    sfL0QG8hdCuBwprev/search3Fq3Dgmo26um3D126hl
    3Den26safe3Dactive26sa3DN26gbv3D226tbm3Di
    schum1itbs1
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