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Microbiology: A Systems Approach, 2nd ed.

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Can be used to cut DNA in to smaller pieces for further study or to remove and insert sequences ... The pieces of DNA produced are called restriction fragments ... – PowerPoint PPT presentation

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Title: Microbiology: A Systems Approach, 2nd ed.


1
Microbiology A Systems Approach, 2nd ed.
  • Chapter 10 Genetic Engineering- A Revolution in
    Molecular Biology

2
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

3
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

4
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

5
Figure 10.1
6
  • 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)

7
Ligase and Reverse Transcriptase
  • Ligase Enzyme necessary to seal sticky ends
    together
  • Reverse transcriptase enzyme that is used when
    converting RNA into DNA

8
Figure 10.2
9
Analysis of DNA
  • Gel electrophoresis produces a readable pattern
    of DNA fragments

Figure 10.3
10
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

11
Figure 10.4
12
Probes Used for Diagnosis
Figure 10.5
13
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

14
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

15
Figure 10.6
16
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

17
Figure 10.7
18
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

19
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

20
Figure 10.8
21
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)

22
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

23
Figure 10.9
24
Characteristics of Cloning Hosts
25
Construction of a Recombinant, Insertion into a
Cloning Host, and Genetic Expression
Figure 10.10
26
Figure 10.11
27
Synthetic Biology Engineering New Genetic
Capabilities
  • Scientists are attempting to create microbes that
    produce hydrogen as fuel
  • Can use recombinant techniques mentioned
    previously

28
10.4 Biochemical Products of Recombinant DNA
Technology
29
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

30
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

31
Transgenic Plants Improving Crops and Foods
  • Agrobacterium can transfect host cells
  • This idea can be used to engineer plants

32
Figure 10.12
33
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34
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

35
Figure 10.13
36
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37
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

38
Figure 10.14
39
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

40
DNA Technology as Genetic Medicine
  • Some diseases result from the inappropriate
    expression of a protein
  • Prevent transcription or translation of a gene

41
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

42
Figure 10.15
43
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

44
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

45
Figure 10.16
46
Microarray Analysis
  • Allows biologists to view the expression of genes
    in any given cell

Figure 10.17
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