Ch 20: DNA Technology

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Ch 20 DNA Technology
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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.

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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

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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
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• 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!

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• Restriction Enzyme animation
• Animation 2
• Cloning a Gene

Bacteria DNA that has DNA from another organism
spliced in to it.
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Gel Electrophoresis

• Method of rapidly analyzing and comparing genomes.

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Restriction Fragment Length Polymorphisms (RFLPs)
Gel Electrophoresis

• The restriction pattern is different for every
  organism.
• This is why you get different banding patterns.

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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

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

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

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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.

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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.

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• 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.

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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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