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

Microbiology: A Systems
Approach, 2nd ed.
Chapter 10: Genetic Engineering- A
Revolution in Molecular Biology
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
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Tools and techniques
Methods in recombinant DNA technology
Biochemical products of recombinant DNA technology
Genetically modified organisms
Genetic treatments
Genome analysis
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
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
Figure 10.1
• 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 lengthsrestriction fragment length polymorphisms
(RFLPs)
Ligase and Reverse Transcriptase
• Ligase: Enzyme necessary to seal sticky ends
together
• Reverse transcriptase: enzyme that is used when
converting RNA into DNA
Figure 10.2
Analysis of DNA
• Gel electrophoresis: produces a readable pattern of
DNA fragments
Figure 10.3
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
Figure 10.4
Probes Used for Diagnosis
Figure 10.5
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
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
Figure 10.6
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
Figure 10.7
Three Basic Steps that Cycle
• Denaturation
– Heat to 94°C to separate in to two strands
– Cool to between 50°C and 65°C
• Priming
– Primers added in a concentration that favors binding to the
complementary strand of test DNA
– Prepares the two strands (amplicons) for synthesis
• Extension
– 72°C
– DNA polymerase and nucleotides are added
– Polymerases extend the molecule
• The amplified DNA can then be analyzed
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
Figure 10.8
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)
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
Figure 10.9
Characteristics of Cloning Hosts
Construction of a Recombinant,
Insertion into a Cloning Host, and
Genetic Expression
Figure 10.10
Figure 10.11
Synthetic Biology: Engineering New
Genetic Capabilities
• Scientists are attempting to create microbes
that produce hydrogen as fuel
• Can use recombinant techniques mentioned
previously
10.4 Biochemical Products of
Recombinant DNA Technology
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
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
Transgenic Plants: Improving Crops
and Foods
• Agrobacterium can transfect host cells
• This idea can be used to engineer plants
Figure 10.12
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
Figure 10.13
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
Figure 10.14
in vivo
• Skips the intermediate step of incubating
excised patient tissue
• Instead the naked DNA or a virus vector is
directly introduced into the patient’s tissues
DNA Technology as Genetic Medicine
• Some diseases result from the inappropriate
expression of a protein
• Prevent transcription or translation of a gene
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
Figure 10.15
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
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
Figure 10.16
Microarray Analysis
• Allows biologists to view the expression of
genes in any given cell
Figure 10.17