Recombinant DNA Technology

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Recombinant DNA Technology

• Combines genes from different sources into a
  single DNA molecule
• May be from different species
• Human Genome Project
• The complete set of genetic material in a human
  as defined by the order of bases in DNA is called
  a genome
• The HGP was completed in 2000

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Genomes

• Genomes that have already been sequenced include
• Fruit fly, bakers yeast, E. coli, rice plant
• Applications include
• Medicine
• Agriculture

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Engineering Bacteria

• Bacteria contain DNA as a small, circular
  molecule called a plasmid
• A plasmid can copy and pass to another bacterial
  cell resulting in gene sharing
• Biologists use plasmids for gene cloning.
• DNA for a desired gene is inserted in a plasmid
  where it makes many copies

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How do Biologists remove a gene from one
DNA molecule and put it in another?
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Restriction Enzymes (Cutting and
Pasting DNA)

• A piece of DNA with the desired gene must be
  cut out of the very long DNA molecule
• The tools used to do so are called restriction
  enzymes
• Each restriction enzyme recognizes a short
  specific sequence and makes a staggered cut
• Eco RI, Bam HI, Hind III, Hae III
• This leaves single-stranded DNA hangs off and is
  called a sticky end which can recombine the DNA

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How do the DNA fragments get put back together?
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Pasting DNA

• The sticky ends from the newly cut DNA are
  available to bind to any sequence that is
  complementary to it.
• The complementary sticky ends of two DNA
  fragments join by base-pairing with each other
• The enzyme DNA ligase pastes the sticky ends
  together to repair the DNA backbone

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Genomic Library

• The complete collection of cloned DNA fragments
  from an organism
• The recombinant plasmids can only contain only a
  few genes
• The entire genome is stored within plasmids

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How does a Biologist find a specific gene from a
genomic library?
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Nucleic Acid Probes

• The biologist must know part of the gene sequence
• Nucleotides are labeled with a radioactive
  isotope to build a complementary single strand of
  DNA
• Called a nucleic acid probe
• The DNA is treated chemically or by heat to
  separate the DNA strands and the probe tags the
  correct portion

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GMO Genetically Modified Organisms

• Transgenic Plants
• Delay ripening
• Nutritional content
• Resistance to disease
• Herbicide resistance

• Transgenic Animals
• Better quality wool
• Leaner meat
• Faster maturation
• Medicinal purpose

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Transgenic Plants
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Animal Cloning

• The nucleus from an egg cell
  is removed
• A new nucleus, genetically altered,
  is inserted into the egg
• The cloned organism will form naturally

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DNA Applications

• PCR
• Gel Electrophoresis
• Genetic Markers
• DNA fingerprinting

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Polymerase Chain Reaction

• A technique that makes many copies of certain
  segment of DNA without using living cells
• Can generate 100 billion copies in 1 hour
• Uses less than 1ug of material
• Using PCR to produce multiple copies makes
  analysis of a sample easier
• Copies of ancient DNA may be cloned from a 40,000
  year old wooly mammoth.
• Can detect viral genes in cells infected with the
  AIDS virus

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Gel Electrophoresis

• Technique used for sorting molecules, or
  fragments, by length
• First, each DNA is cut up using restriction
  enzymes
• A few drops of each sample are placed in a well
  at one end of a gel (a slab of agarose)
• A charge is run through the gel so that the DNA
  molecules move down the gel.
• The shorter fragments move easier and travel
  farther
• The longer fragments are shorter

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• The gel is then treated with a stain
• The stain makes DNA visible under UV light
• Each band consists of DNA of a specific size
• Each sample has a unique pattern

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How does a Biologist find any differences
in the banding patterns between individual
samples ?
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Genetic Markers

• There are specific portions of the genome called
  genetic markers
• Genetic markers are stretches of DNA that are
  variable among individuals
• DNA fragments may contain certain disease alleles
  for example.
• Radioactively labeled DNA probes can tag the
  bands that contain these markers

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DNA Fingerprinting

• Most organisms have a particular banding pattern
  DNA produced by restriction fragments called a
  DNA Fingerprint
• Markers can be located on the noncoding stretches
  of the human genome
• These markers are unlikely to be shared by other
  individuals
• Can be made from cells in a single drop of blood,
  hair follicle, etc.

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