Analyzing and Engineering Genes

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Analyzing and Engineering Genes

• Chapter 19

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Analyzing and Engineering DNA

• Genetic engineering- Manipulation of DNA
  sequences in organisms
• Recombinant DNA technology used to engineer genes
• Biotechnology-the manipulation of organisms to
  create products or cure diseases
• Goals of genetic engineering
• Improve our understanding of how genes work
• Advance biotechnology

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An ExampleUsing Recombinant DNA Techniques to
Manufacture Proteins The Effort to Cure
Pituitary Dwarfism
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Pituitary Dwarfism

• Pituitary dwarfism results from the abnormal
  production of growth hormone, encoded by the GH1
  gene
• Humans affected by pituitary dwarfism grow slowly
• maximum adult height of about 4 feet
• Could be treated successfully with growth hormone
  therapy, but only if the protein came from humans

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Pituitary Dwarfism

• Using recombinant DNA technology to produce a
  safe supply of growth hormone
• Involved cloning the human gene, introducing the
  gene into bacteria (or yeast), and having these
  microbes synthesize the hormone
• Reverse transcriptase was used to make
  complementary DNA (cDNA) from mRNA isolated from
  pituitary cells

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Cloning the Gene

• Genetic cloningthe process of producing many
  identical copies of a genewas used to clone the
  cDNAs for analysis to determine which encoded the
  growth hormone protein

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Bacteria as a Tool for Manipulating DNA

• In nature, bacteria can transfer DNA in three ways

• Transformation, the taking up of DNA from the
  fluid surrounding the cell
• Can even take up DNA from dead cells

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Bacteria as a Tool for Manipulating DNA

• Conjugation, the union of cells and the DNA
  transfer between them

• Transduction, the transfer of bacterial genes by
  a phage

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• The transferred DNA is then integrated into the
  recipient cells chromosome

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Bacterial Phages as Carriers

• Bacterial plasmids can serve as carriers for gene
  transfer
• An F factor is a DNA segment in bacteria that
  enables conjugation and contains an origin of
  replication
• The F factor starts replication and transfers
  part of the chromosome

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• An F factor can exist as a plasmid, a small
  circular DNA molecule separate from the bacterial
  chromosome
• R plasmids carry genes for resistance of
  antibiotics and that is how bacteria can become
  resistant

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Plasmids are used to customize bacteria

• Plasmids are key tools for DNA technology
• Researchers use plasmids to insert genes into
  bacteria
• Plasmids are obtained from other bacteria
• Desired DNA inserted into plasmid
• Bacteria takes up DNA
• Can be used for several applications

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What are Plasmids?

• Small, circular DNA molecules that replicate
  independently of the chromosome
• Can be used to carry recombinant genes in
  bacteria.
• Restriction endonucleases are enzymes that cut
  DNA at specific base sequences called recognition
  sites
• Often make staggered cuts in the DNA, resulting
  in sticky ends.
• Plasmids and cDNAs cut with the same restriction
  endonuclease can be spliced together at their
  sticky ends

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Transformation Introducing Recombinant Plasmids
into Bacterial Cells
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Transformation

• Plasmids serve as a vectora vehicle for
  transferring recombinant genes to a new host.
• Plasmids can be introduced into bacteria by
  transformation
• Can be used to replicate and make more DNA
• Can be used to make proteins

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Genomic Libraries

• Recombinant DNA
• technology allows the construction of genomic
  libraries
• Genomic libraries
• are sets of DNA
• fragments containing
• all of an organisms genes
• Copies of DNA fragments can be stored in a cloned
  bacterial plasmid or phage

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Using Nucleic Acid Hybridization to Finda Target
Gene
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Nucleic Acid Probes

• A probe is a single-stranded fragment of a
  labeled, known gene
• Binds to a complementary sequence in the sample
  being analyzed
• Can be used to screen for bacterial colonies
  containing a plasmid with the growth hormone gene

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Mass Producing the Gene
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Mass Producing the Growth Hormone

• The human growth hormone cDNA was cloned in a
  plasmid under the control of a bacterial promoter
• Bacteria carrying these plasmids can make a large
  quantity of human growth hormone
• Hormone can be purified and used to treat patients

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Ethical Issues

• Hormone used for kids who did not suffer from
  pituitary dieases
• Approved use only for children projected to reach
  adult heights of less than 5'3" for males and
  less than 4'11" for females.
• Also problems with these hormones being used as
  popular performance-enhancing drug for athletes

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Sequencing DNA
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DNA Sequencing

• Can be used to infer the amino acid sequence of
  the protein product
• Known sequence can be compared to the sequences
  of genes that have the same function in various
  species
• Can be used to infer the function of the protein
• Huge libraries of information about DNA
  sequences, GenBank

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PCR

• Polymerase chain reaction (PCR) is an in vitro
  DNA synthesis reaction in which a section of DNA
  is amplified millions of times

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PCR

• PCR ingredients a DNA template, two primers that
  bracket the region to be amplified, dNTPs,
  buffer, and DNA polymerase.
• Requires about 30 cycles, with each cycle
  containing three steps
• Denaturation to separate the DNA strands,
• Annealing to allow the flanking primers to anneal
  to the denatured DNA
• Extension step for synthesis of the complementary
  strand.

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Gel Electrophoresis

• Physically sorts out macromolecules (DNA, RNA) on
  the basis of their charge and size
• Current is run through the gel and since DNA is
  negatively charged it moves through the gel
• The longer the DNA molecules are. The slower they
  move
• Bands are made, each consisting of DNA molecules
  of one size

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Gel Electrophoresis

• Restriction fragments of DNA can be sorted by size

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Restriction Fragment Analysis

• Everyones DNA sequence is different
• Scientists can compare DNA sequences of different
  individuals based on the size of the fragments
  created by restriction enzymes
• They can only use DNA that varies from person to
  person
• When run on a gel it makes a distinct pattern

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Restriction Fragment Analysis
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Detecting Harmful Alleles

• Radioactive single-stranded DNA complimentary
  strands are used to verify the presence of
  certain nucleic acid sequences known to code for
  harmful alleles

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Gene Therapy

• Gene therapy is the introduction of a gene to
  replace or augment a mutant gene that is causing
  an abnormal phenotype
• The two primary vectors for introducing
  therapeutic genes into human cells are
  retroviruses and adenoviruses
• Adenoviruses only work for a short time
• Retroviruses integrate and are better

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Gene Therapy

• Gene therapy is highly experimental, extremely
  expensive, and very controversial.
• Holds great promise for the treatment of a wide
  variety of inherited diseases
• Require many years of additional research and
  testing, as well as the refinement of legal and
  ethical guidelines.

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Biotechnology

• Genetic engineering in agriculture focuses on
• Reducing herbivory
• Making crops herbicide resistant
• Improving the quality of the food product

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Golden Rice

• Half the world's population depends on rice as a
  staple food
• Contains no vitamin A.
• Lack of vitamin A in the diet leads to blindness,
  diarrhea, respiratory infections, and childhood
  diseases such as measles.

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Agrobacterium tumefaciens

• Agrobacterium tumefaciens is often used for
  genetic transformation of plants through transfer
  of its Ti (tumor-inducing) plasmid
• Can be disarmed and used to insert DNA

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Golden Rice

• ß-carotene is a precursor of vitamin A.
• Golden rice has been genetically modified to
  contain beta-carotene
• This rice could help prevent vitamin A deficiency

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GMOs and the Environment

• Genetic engineering involves some risks
• Possible ecological damage from pollen transfer
  between GM and wild crops
• Pollen from a transgenic variety of corn that
  contains a pesticide may stunt or kill monarch
  caterpillars