Microbiology: A Systems Approach, 2nd ed.

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Microbiology A Systems Approach, 2nd ed.

• Chapter 10 Genetic Engineering- A Revolution in
  Molecular Biology

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10.1 Basic Elements and Applications of Genetic
Engineering

• Basic science when no product or application is
  directly derived from it
• Applied science useful products and
  applications that owe their invention to the
  basic research that preceded them
• Six applications and topics in genetic
  engineering
• Tools and techniques
• Methods in recombinant DNA technology
• Biochemical products of recombinant DNA
  technology
• Genetically modified organisms
• Genetic treatments
• Genome analysis

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10.2 Tools and Techniques of Genetic Engineering

• DNA The Raw Material
• Heat-denatured DNA
• DNA strands separate if heated to just below
  boiling
• Exposes nucleotides
• Can be slowly cooled and strands will renature

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Restriction Endonucleases

• Enzymes that can clip strands of DNA crosswise at
  selected positions
• Hundreds have been discovered in bacteria
• Each has a known sequence of 4 to 10 pairs as its
  target
• Can recognize and clip at palindromes

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Figure 10.1
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• Can be used to cut DNA in to smaller pieces for
  further study or to remove and insert sequences
• Can make a blunt cut or a sticky end
• The pieces of DNA produced are called restriction
  fragments
• Differences in the cutting pattern of specific
  restriction endonucleases give rise to
  restriction fragments of differing lengths-
  restriction fragment length polymorphisms (RFLPs)

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Ligase and Reverse Transcriptase

• Ligase Enzyme necessary to seal sticky ends
  together
• Reverse transcriptase enzyme that is used when
  converting RNA into DNA

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Figure 10.2
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Analysis of DNA

• Gel electrophoresis produces a readable pattern
  of DNA fragments

Figure 10.3
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Nucleic Acid Hybridization and Probes

• Two different nucleic acids can hybridize by
  uniting at their complementary regions
• Gene probes specially formulated
  oligonucleotide tracers
• Short stretch of DNA of a known sequence
• Will base-pair with a stretch of DNA with a
  complementary sequence if one exists in the test
  sample
• Can detect specific nucleotide sequences in
  unknown samples
• Probes carry reporter molecules (such as
  radioactive or luminescent labels) so they can be
  visualized
• Southern blot- a type of hybridization technique

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Figure 10.4
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Probes Used for Diagnosis
Figure 10.5
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Fluorescent in situ Hybridizaton (FISH)

• Probes applied to intact cells
• Observed microscopically for the presence and
  location of specific genetic marker sequences
• Effective way to locate genes on chromosomes

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Methods Used to Size, Synthesize, and Sequence DNA

• Relative sizes of nucleic acids usually denoted
  by the number of base pairs (bp) they contain
• DNA Sequencing Determining the Exact Genetic
  Code
• Most detailed information comes from the actual
  order and types of bases- DNA sequencing
• Most common technique Sanger DNA sequence
  technique

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Figure 10.6
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Polymerase Chain Reaction A Molecular Xerox
Machine for DNA

• Some techniques to analyze DNA and RNA are
  limited by the small amounts of test nucleic acid
  available
• Polymerase chain reaction (PCR) rapidly increases
  the amount of DNA in a sample
• So sensitive- could detect cancer from a single
  cell
• Can replicate a target DNA from a few copies to
  billions in a few hours

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Figure 10.7
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Three Basic Steps that Cycle

• Denaturation
• Heat to 94C to separate in to two strands
• Cool to between 50C and 65C
• Priming
• Primers added in a concentration that favors
  binding to the complementary strand of test DNA
• Prepares the two strands (amplicons) for
  synthesis
• Extension
• 72C
• DNA polymerase and nucleotides are added
• Polymerases extend the molecule
• The amplified DNA can then be analyzed

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10.3 Methods in Recombinant DNA Technology

• Primary intent of recombinant DNA technology-
  deliberately remove genetic material from one
  organism and combine it with that of a different
  organism
• Form genetic clones
• Gene is selected
• Excise gene
• Isolate gene
• Insert gene into a vector
• Vector inserts DNA into a cloning host

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Figure 10.8
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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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10.4 Biochemical Products of Recombinant DNA
Technology
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10.5 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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10.6 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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10.7 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

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Figure 10.16
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Microarray Analysis

• Allows biologists to view the expression of genes
  in any given cell

Figure 10.17