Technical Aspects of Recombinant DNA and Gene Cloning

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Technical Aspects of Recombinant DNA and Gene
Cloning

• Strategies for obtaining genes in an isolated
  state
• DNA removed from cells, separated into fragments,
  inserted into a vector, and cloned then undergo
  Southern blotting and probed
• Gene can be synthesized from isolated mRNA
  transcripts
• Gene can be amplified using PCR
• Once isolated, genes can be maintained in a
  cloning host and vector (genomic library)

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Characteristics of Cloning Vectors

• Capable of carrying a significant piece of the
  donor DNA
• Readily accepted by the cloning host
• Must have a promoter in front of the cloned gene
• Vectors (such as plasmids and bacteriophages)
  should have three important attributes
• An origin of replication somewhere on the vector
• Must accept DNA of the desired size
• Contain a gene that confers drug resistance to
  their cloning host

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Figure 10.9
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Characteristics of Cloning Hosts
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Construction of a Recombinant, Insertion into a
Cloning Host, and Genetic Expression
Figure 10.10
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Figure 10.11
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Synthetic Biology Engineering New Genetic
Capabilities

• Scientists are attempting to create microbes that
  produce hydrogen as fuel
• Can use recombinant techniques mentioned
  previously

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Biochemical Products of Recombinant DNA Technology
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Genetically Modified Organisms

• Transgenic or genetically modified organisms
  (GMOs) recombinant organisms produced through
  the introduction of foreign genes
• These organisms can be patented

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Recombinant Microbes Modified Bacteria and
Viruses

• Genetically altered strain of Pseudomonas
  syringae
• Can prevent ice crystals from forming
• Frostban to stop frost damage in crops
• Strain of Pseudomonas fluorescens
• Engineered with a gene from Bacillus
  thuringiensis
• Codes for an insecticide
• Drug therapy
• Bioremediation

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Transgenic Plants Improving Crops and Foods

• Agrobacterium can transfect host cells
• This idea can be used to engineer plants

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Figure 10.12
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Transgenic Animals Engineering Embryos

• Several hundred strains have been introduced
• Can express human genes in organs and organ
  systems
• Most effective way is to use viruses

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Figure 10.13
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Genetic Treatments Introducing DNA into the Body

• Gene Therapy
• For certain diseases, the phenotype is due to the
  lack of a protein
• Correct or repair a faulty gene permanently so it
  can make the protein
• Two strategies
• ex vivo
• in vivo

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Figure 10.14
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in vivo

• Skips the intermediate step of incubating excised
  patient tissue
• Instead the naked DNA or a virus vector is
  directly introduced into the patients tissues

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DNA Technology as Genetic Medicine

• Some diseases result from the inappropriate
  expression of a protein
• Prevent transcription or translation of a gene

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Antisense DNA and RNA Targeting Messenger RNA

• Antisense RNA bases complementary to the sense
  strand of mRNA in the area surrounding the
  initiation site
• When it binds to the mRNA, the dsRNA is
  inaccessible to the ribosome
• Translation cannot occur
• Single-stranded dNA usually used as the antisense
  agent (easier to manufacture)
• For some genes, once the antisense strand bound
  to the mRNA, the hybrid RNA was not able to leave
  the nucleus
• Antisense DNA when delivered into the cytoplasm
  and nucleus, it binds to specific sites on any
  mRNAs that are the targets of therapy

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Figure 10.15
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Genome Analysis Maps, Fingerprints, and Family
Trees

• Possession of a particular sequence of DNA may
  indicate an increased risk of a genetic disease
• Genome Mapping and Screening An Atlas of the
  Genome
• Locus the exact position of a particular gene
  on a chromosome
• Alleles sites that vary from one individual to
  another the types and numbers are important to
  genetic engineers
• Mapping the process of determining location of
  loci and other qualities of genomic DNA
• Linkage maps show the relative proximity and
  order of genes on a chromosome
• Physical maps more detailed arrays that also
  give the numerical size of sections in base pairs
• Sequence maps produced by DNA sequencers
• Genomics and bioinformatics managing mapping
  data

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DNA Fingerprinting A Unique Picture of a Genome

• DNA fingerprinting tool of forensic science
• Uses methods such as restriction endonucleases,
  PCR, electrophoresis, hybridization probes, and
  Southern blot technique