Genetic Technology

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Genetic Technology
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Biotechnology

• Traditional vs. Modern Biotechnology
• History of Biotechnology
• Ethical issues

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Genetic Engineering

• Involves cutting (or cleaving) DNA from one
  organism into small fragments and inserting the
  fragments into a host organism of the same or a
  different species
• AKA Recombinant DNA technology
• Recombinant DNA is made by connecting, or
  recombining, fragments of DNA from different
  sources

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Transgenic Organisms

• Contain recombinant DNA
• Examples Glowing tobacco (pg 349)
• Bt corn
• Golden rice

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Steps involved in creating a transgenic organism

• Step 1 Isolate the DNA fragment that will be
  inserted (use a restriction enzyme)
• Step 2 attach the DNA fragment to the vehicle
  (virus or bacteria DNA) by gene splicing
• Step 3 transfer the vehicle into the host
  organism (recombined DNA is transferred to a
  bacterial cell)

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Uses of Recombinant DNA

• Industry
• Medicine
• Transgenic animals
• Agriculture
• Transgenic plants

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A Revolution in Biological Science

• In mid-1970s, newly developed recombinant DNA
  technology provided, for the first time, powerful
  techniques for studying and manipulating DNA
• Recombinant DNA technology allows biologists to
  redirect the genetic activity of organisms
• Techniques and approaches include
• Splicing Specific genes can be added or removed
  by cutting and rearranging DNA
• Genetic engineering Modification of the DNA of
  an organism to produce new genes
• DNA cloning Large quantities of DNA can be
  obtained relatively easily by cloning cells and
  amplifying specific DNA sequences, both in situ
  (inside) and in vitro (out of) the cell
• Biotechnology, the overall corporate-driven use
  of genetic engineering, which already has had
  great impact on our lives

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History and Terminology

• Biotechnology has its roots in microbiology the
  study of micro- organisms (usually bacteria)
• Bacteriophage (viruses of bacteria) were first
  used to try to understand how DNA worked (recall
  Hershey-Chase)
• Scientists learned how to make bacteria competent
  for transformation (recall Griffith) by
  modification of the ionic environment
• Made the cell wall more permeable
• Allowed the cells to take up DNA
• Genetic engineering not possible prior to
  discovery of restriction endonucleases
  (restriction enzymes) by Ham Smith and Daniel
  Nathans (Johns Hopkins Nobel Prize winners
  1978)
• Specifically clip strands of DNA
• Many different types

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Restriction Enzymes 1

• Restriction enzymes are natural, bacterial
  molecular scissors normally used to destroy
  non-host (such as bacteriophage) DNA
• Cut DNA at specific base pair sequences many are
  palindromic
• A linguistic palindrome has the same
  informational sequence forward and backward
• MadamImadam is a linguistic palindrome
• A nucleic acid palindrome has the same sequence
  on two antiparallel, complementary,
  hydrogen-bonded strands
• e.g. AACGTT will pair with TTGCAA these are
  palindromes
• Restriction enzymes cut at the ends of the
  palindromic sequences (red in the example here)
• They are cut in a staggered fashion

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Restriction Enzymes 2

• Restriction enzymes snip the phosphodiester bond
  at a very VERY specific location based on the
  sequence information
• Staggered cuts on ends of palindromic regions
  leave strands with complementary sticky ends
  when separated (usuallywith heat)
• Segments of DNA with sticky ends can hydrogen
  bond with complementary sequences
• The open spaces can be joined with purified
  naturally-occurring DNA ligases ( ) process
  is called splicing

Heat denaturation
L
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Vector DNA

• A vector is a genome that carries foreign DNA
  into a host cell
• Used to transform competent (can take up DNA)
  bacteria
• Bacteriophage (bacterial viruses recall
  Hershey-Chase)
• Can carry DNA segments of up to 15kb
• Engineered mammalian viruses used in mammalian
  cells
• DNA incorporated into nuclear DNA of mammalian
  cell
• Plasmids are small rings of double-stranded DNA
  that commonly occur in bacteria
• Can carry DNA segments lt 10kb in size 1Kb1000
  bps
• Often carry genes for resistance to antibiotics
• Can be used to provide a selectable marker which
  allows only transformed cells to live. Cells
  containing ampicillin resistance gene inserted by
  transformation can be grown on ampicillin-rich
  media nontransformed cells die

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Plasmids and Bacteria
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Recombinant DNA Is Formedby Splicing DNA From a
VectorInto Host DNA
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PCR Is Used to Amplify DNA in Vitro

• The Polymerase Chain Reaction (PCR) allows
  amplification of a small amount of targeted DNA
  in a short time. It is very simple but very
  powerful.
• PCR has three steps
• Denaturation. A buffered mixture of primers,
  nucleotides, Taq polymerase and DNA fragments is
  heated to dissociate ds DNA into ssDNA
• Annealing of primers. The solution is cooled and
  the primers bind to complementary sequences of
  the DNA at the ends
• Primer Extension. DNA polymerase then uses the
  nucleotides to extend and make more copies of
  each strand
• The process is repeated over and over to produce
  millions of copies of the original DNA strand

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PCR
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PCR Characteristics

• DNA Taq polymerase isolated from the thermophilic
  bacterium Thermus aquaticus is used as it is not
  damaged by the heat
• After 20 cycles a single fragment produces more
  than one million (220) copies
• 30 cycles will produce a billion times the
  original amount (230), enough amplification to
  reveal the presence of a single copy of a
  specific target sequence
• The use of PCR is virtually limitless
• Criminal investigations (DNA fingerprints) from a
  speck of blood or single hair
• Detection of genetic defects in very early
  embryos by collecting a few sloughed-off cells
  from the amniotic fluid (amniocentesis) and
  amplifying the DNA
• Used to examine historical figures and extinct
  species such as mammoths and dodos
• Very sensitive and samples easily contaminated

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Gel Electrophoresis Is Widely Used to Separate
DNA and RNA

• DNA and RNA are negatively charged, and move
  through a gel at varying speeds due to different
  molecular lengths (sizes)
• Restriction endonucleases can be used to clip the
  DNA
• DNA fragments are loaded on a gel an electric
  field is applied
• Bigger DNA fragments migrate through the gel more
  slowly than small fragments
• Fragments can be stained and visualized migrating
  as bands under UV light
• DNA fragments can be transferred (blotted) to a
  filter, denatured and incubated with a
  radioactive or fluorescent probe which will
  hybridize to the target sequence and be revealed
  by autoradiography or sensitive color digital
  camera

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Southern and Northern Blotting

• Blots for DNA are called Southern blots
• Named after its inventor, E.M. Southern (1975)
• DNA is separated on a gel
• Gel is transferred onto nitrocellulose or a nylon
  membrane
• Membrane is incubated with radioactive ssDNA
  probe of the gene of interest
• Probe hybridizes to the blot where there is a
  fragment with a complementary sequence
• The radioactive bands on the blot identify
  fragments of interest
• If RNA blotted, called Northern blot

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Western Blotting

• Proteins separated in a gel
• Proteins blotted onto a membrane
• Antibodies specific for a particular protein are
  applied
• Antibodies stick to target proteins ONLY
• Revealed by additional antibodies attached to
  enzymes that precipitate a colored product

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DNA Sequences Contain Much Information

• Can determine the actual protein encoding
  regions the ORFs
• Regions containing transcriptional signals and
  RNA processing can be recognized
• Amino acid sequences of proteins can be inferred
  from the base sequence much faster and easier
  than from the protein directly
• Reveals structure of chromosomes, possibly
  helpful in determining evolution, phylogenies,
  and fighting disease

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DNA Nucleotide Sequencing

• Radioactive DNA is replicated off the host
  template DNA
• Dideoxynucleotides (ddNTPs lacking OH at 3 and
  2) are incorporated in small quantities in the
  reaction mixture to label sequences which contain
  those deoxybases (the ddNTPs jam DNA polymerase)
• Reaction mixtures contain DNA polymerase,
  radioactive primers, single-stranded DNA
  fragment, 4 deoxynucleotides. Four tubes are
  prepared each containing a different
  dideoxynucleotide (ddATP, ddCTP, ddGTP or ddTTP)
• Fragments of varying length are formed in each
  mixture the end points occur at the 4 different
  ddNTP
• Fragments are separated based on length by
  electrophoresis
• Autoradiography reveals the presence of the
  radioactively-labeled DNA fragments
• The DNA sequence is literally read off of the
  gel, using the 4 lanes derived from the 4
  reaction tubes.

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Restriction Fragment LengthPolymorphism Analysis

• Each individual carries with it a record of the
  variation in genetic organization from its
  previous generations. There is a LOT of variation
  in individuals of a population
• Restriction enzymes are used to cut DNA into
  fragments
• The fragments are of different lengths in
  different individuals since each host DNA is
  unique. When three different individuals DNA are
  cut with a restriction endonuclease, 3 different
  fragment sizes are likely to be produced, unless
  they are identical triplets

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An RFLP Autoradiogram

• A DNA fingerprint produced by gel
  electrophoresis reveals different banding
  patterns restriction fragment length
  polymorphisms (RFLPs)
• This technology is particularly important in
  determination of paternity and in forensics
• Here, M mother, F father, and C children.
  Note that children have all bands of M and F
  lanes.