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Ch 20: DNA Technology

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Title: Ch 20: DNA Technology


1
Ch 20 DNA Technology
2
Introduction
  • The mapping and sequencing of the human genome
    has been made possible by advances in DNA
    technology.
  • Human genome project
  • We are now developing techniques for making
    recombinant DNA
  • Genes from two different sources - often
    different species - are combined into the same
    molecule.
  • DNA technology has launched a revolution in
    biotechnology.
  • The manipulation of organisms or their components
    to make useful products.
  • DNA technology is now applied in areas ranging
    from agriculture to criminal law, but its most
    important achievements are in basic research.

3
Recombinant DNA
  • Recombinant DNA
  • Taking DNA from two sources and combining then
    into one molecule.
  • Occurs naturally in viral transduction, bacterial
    transformation, and conjugation
  • Biotechnology (genetic engineering)
  • Engineering genes in the Lab

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Many tools and techniques have been developed to
manipulate and engineer genes.
  • Restriction Enzymes
  • Gel Electrophoresis
  • -Restriction Fragment Polymorphisms
  • DNA Probe
  • Polymerase Chain Reaction
  • Complementary DNA

5
Restriction Enzymes
  • Discovered in the 1960s
  • Extracted from bacteria
  • Used to fend off bacteriophages
  • Appear to serve a host-defense role
  • Protect own DNA by methaylation of adenines
    cytosines
  • REs cut DNA at specific sites called recognition
    sequences or sites.
  • Leaving fragments of DNA
  • Scientists have isolated 100s of REs
  • Named for the bacteria in which they were found.
  • EcoRI
  • BamHI
  • HindIII

clip
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Naming
Restriction enzymes are named based on the
bacteria in which they are isolated in the
following manner
E Escherichia (genus)
co coli (species)
R RY13 (strain)
I First identified Order ID'd in bacterium
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Enzyme Organism from which derived Target sequence(cut at )5' --gt3'
Ava I Anabaena variabilis C C/T C G A/G G
Bam HI Bacillus amyloliquefaciens G G A T C C
Bgl II Bacillus globigii A G A T C T
Eco RI Escherichia coli RY 13 G A A T T C
Eco RII Escherichia coli R245 C C A/T G G
Hae III Haemophilus aegyptius G G C C
Hha I Haemophilus haemolyticus G C G C
Hind III Haemophilus inflenzae Rd A A G C T T
Hpa I Haemophilus parainflenzae G T T A A C
Kpn I Klebsiella pneumoniae G G T A C C
Mbo I Moraxella bovis G A T C
Mbo I Moraxella bovis G A T C
Pst I Providencia stuartii C T G C A G
Sma I Serratia marcescens C C C G G G
SstI Streptomyces stanford G A G C T C
8
  • The restriction sites are often a symmetrical
    series of four to eight bases on both strands
    running in opposite directions.
  • If the restriction site on one strand is
    3-CTTAAG-5, the complementary strand is
    5-GAATTC-3.
  • Because the target sequence usually occurs (by
    chance) many times on a long DNA molecule, an
    enzyme will make many cuts.
  • Copies of a DNA molecule will always yield the
    same set of restriction fragments when exposed to
    a specific enzyme.

CLIP
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  • Restriction enzymes cut covalent phosphodiester
    bonds of both strands
  • often in a staggered way creating single-stranded
    ends, sticky ends.
  • These extensions will form hydrogen-bonded base
    pairs with complementary single-stranded
    stretches on other DNA molecules cut with the
    same restriction enzyme.
  • These DNA fusions can be made permanent by DNA
    ligase which seals the strand by catalyzing the
    formation of phosphodiester bonds.

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Must use same restriction enzyme on both.
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  • Setting up a simple restriction digestion
  • DNA Reliable cleavage by restriction enzymes
    requires DNA that is free from contaminants such
    as phenol or ethanol. Excessive salt will also
    interfere with digestion by many enzymes,
    although some are more tolerant of that problem.
  • An appropriate buffer Different enzymes cut
    optimally in different buffer systems, due to
    differing preferences for ionic strength and
    major cation. When you purchase an enzyme, the
    company almost invariably sends along the
    matching buffer as a 10X concentrate.
  • The restriction enzyme!

13
  • Restriction Enzyme animation
  • Animation 2
  • Cloning a Gene

Bacteria DNA that has DNA from another organism
spliced in to it.
14
Gel Electrophoresis
  • Method of rapidly analyzing and comparing genomes.

15
Restriction Fragment Length Polymorphisms (RFLPs)
Gel Electrophoresis
  • The restriction pattern is different for every
    organism.
  • This is why you get different banding patterns.

16
Agar is an unbranched polysaccharide obtained
from the cell walls of some species of red algae
or seaweed
  • DNA fragments are visualized by staining with
    ethidium bromide. This fluorescent dye
    intercalates between bases of DNA and RNA

17
  • Rate of movement depends on size, electrical
    charge, and other physical properties of the
    macromolecules.

18
  • Separation depends mainly on size (length of
    fragment) with longer fragments migrating less
    along the gel.

19
Simulation
  • Because DNA has a negative charge, it moves
    toward the opposite side.
  • Smaller fragments move greater distances

Animation
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We can tie together several molecular techniques
to compare DNA samples from three individuals
  • We start by adding the restriction enzyme to each
    of the three samples to produce restriction
    fragments.
  • We then separate the fragments by gel
    electrophoresis.
  • Southern blotting (Southern hybridization) allows
    us to transfer the DNA fragments from the gel to
    a sheet of nitrocellulose paper, still separated
    by size.
  • Southern blotting -method in molecular biology of
    enhancing the result of an agarose gel
    electrophoresis by marking specific DNA sequences
  • This also denatures the DNA fragments.
  • Bathing this sheet in a solution containing our
    probe allows the probe to attach by base-pairing
    (hybridize) to the DNA sequence of interest and
    we can visualize bands containing the label with
    autoradiography.

Animation
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Polymerase Chain Reaction (PCR) Making copies
  • Common method of creating copies of specific
    fragments of DNA
  • PCR rapidly amplifies a single DNA molecule into
    many billions of molecules

Animation
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Complementary DNA (cDNA)
  • When scientists clone a human gene in a
    bacterium, the introns present a problem.
  • Bacteria lack introns and have no way to cut them
    out.

Animation
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Complementary DNA (cDNA)
  • In order to clone a human gene in a bacterium,
    the introns need to be removed.
  • The genes is allowed to be transcribed and fully
    processed into mRNA.
  • Reverse transcriptase is added to the mRNA and
    DNA copies are made.
  • The DNA made by this process is called cDNA.

25
DNA probe DNA Tagging
  • A DNA probe is a radioactively labeled single
    strand of nucleic acid molecule used to tag a
    specific sequence in a DNA sample.
  • The probe bonds to a complementary sequence
    wherever it occurs.
  • Can identify genetic defects

Animation
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Gene Therapy
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Cloning
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Genetically Modified Organisms
Transgeneic Organisms CLIP
  • Clip

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Transgenic Organisms
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Transgenic Tobacco, from 1986. This is an
ordinary photographic image of a tobacco plant
engineered to express a firefly gene which
produces luciferase.
CLIP
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Plasmids as vectors
  • Lab on Fri
  • Animation

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Golden Rice 23 times more Vitamin A
Called a Transgenic Organism
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  • Researchers use recombinant DNA technology to
    analyze genetic changes.
  • They cut, splice together, and insert the
    modified DNA molecules from different species
    into bacteria or another type of cell that
    rapidly replicates and divides.
  • The cells copy the foreign DNA right along with
    their own DNA.
  • An example of this is the gene for human insulin
    inserted into a bacterium. This is how human
    insulin is mass produced.

33
  • Not only does genetic engineering have
    applications in medicine and the environment, it
    also has uses in industry and agriculture.
  • Sheep are used in the production of alpha-1
    antitrypsin, which is used in the treatment of
    emphysema.
  • Goats are also producing the CFTR protein used in
    the treatment of cystic fibrosis.

34
In the plant world, the buds of cotton plants are
vulnerable to worm attacks. The buds of a
modified cotton plant resist these worms,
resulting in increased cotton production. These
gene insertions are ecologically safer than
pesticides. They affect only the targeted pest.
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Plant biologists have used DNA technology to
produce plants with many desirable traits. These
include increased disease resistance, herbicide
resistance, and increased nutritional content.
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  • Scientists today have developed genetically
    altered bacteria.
  • Among them are strains of bacteria that
  • eat up oil spills
  • manufacture alcohol and other chemicals
  • process minerals.
  • There is concern about possible risks to the
    environment and the general population as
    genetically engineered bacteria are introduced.

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