Download Foundations in Microbiology

Chapter 10
Genetic Engineering:
A Revolution in
Molecular Biology
1
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Genetic Engineering
• Basic knowledge is used to derive applied science
or useful products
• Direct, deliberate modification of an organism’s
genome
– Bioengineering
• Biotechnology – use of an organism’s
biochemical and metabolic pathways for industrial
production
2
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Tools and Techniques of
Genetic Engineering
Practical Properties of DNA
• Intrinsic properties of DNA hold true even in a test tube
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• DNA heated from 90°C to
95°C; the two strands
separate. The nucleotides
can be identified, replicated,
or transcribed.
Heating
Cooling
• Slowly cooling the DNA
allows complementary
(a) DNA heating and cooling. DNA responds to heat by denaturing—losing
nucleotides to hydrogen
its hydrogen bonding and thereby separating into its two strands. When
cooled, the two strands rejoin at complementary regions. The two strands
bond and the DNA will
need not be from the same organisms as long as they have matching sites.
regain double-stranded form
3
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Enzymes for Dicing, Splicing, and
Reversing Nucleic Acids
Restriction endonucleases – recognize specific sequences
of DNA and break phosphodiester bonds between
adjacent nucleotides
•
The enzymes can be used to cleave DNA at desired sites
•
Recognize and clip the DNA at palindrome base
sequences
•
Used in the lab to cut DNA into smaller pieces –
restriction fragments
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Endonuclease
Cutting
pattern
EcoRI
G A A T T C
C T T A A G
HindIII
HaeIII
A A G C T T
T T C G A A
G G C C
C C G G
(b) Examples of endonucleases, palindromes and cutting patterns.
The first two are staggered cuts, and the third is a blunt cut.
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4
Restriction Fragment Length
Polymorphisms
•
•
DNA sequences vary, even among members of
the same species
Differences in the cutting pattern of specific
restriction endonucleases give rise to fragments
of differing lengths (RFLPs)
5
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Enzymes for Dicing, Splicing, and
Reversing Nucleic Acids
•
Ligase – rejoins phosphate-sugar bonds
(sticky ends) cut by endonucleases
•
Used for final splicing of genes into plasmids
and chromosomes
6
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Copyright © McGraw-Hill Education. Permission required for reproduction or display.
Restriction endonuclease
makes staggered cut
at palindrome.
Site of cut
(1)
C TAG
GAT C
TAG
C
C
GAT
Sticky ends
(2)
DNA
Organism
1
DNA
Organism
2
Action of restriction endonucleases. (1) A restriction endonuclease recognizes and cleaves DNA at the site of
a specific palindromic sequence. Cleavage usually produces staggered tails called sticky ends that accept
complementary tails for gene splicing. This palindrome is cut by Aci I.
(2) The sticky ends can be used to join DNA from different organisms by cutting it with the same restriction
enzyme, ensuring that both fragments have two complementary ends. This palindrome is cut by Taq I.
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7
Enzymes for Dicing, Splicing, and
Reversing Nucleic Acids
•
Reverse transcriptase – makes a DNA copy of RNA –
cDNA
•
cDNA can be made from mRNA, tRNA, or rRNA
•
Provides a means of synthesizing eukaryotic genes from
mRNA transcripts – synthesized gene is free of introns
8
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9
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Methods for Analysis of DNA
• Gel electrophoresis - separates DNA fragments based
on size
– DNA samples are placed on soft agar gel and
subjected to an electric current
10
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Methods for Analysis of DNA
– Negative charge of molecule causes DNA to move
toward positive pole
– Rate of movement is dependent on size of fragment –
larger fragments move more slowly
11
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Methods for Analysis of DNA
– Fragments are stained for observation
– Useful in characterizing DNA fragments and comparing
for genetic similarities
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Sample
Sample
1
2
3
4
Known
DNA
Size
Markers
Sample
Well
Sample
Result, following development
5
No. of base
pairs in band
5160
5035
4910
3160
2910
2760
2260
1510
1260
1010
12
(b)
© Kathy Park Talaro
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Concept Check:
DNA fragments move through the gel during
electrophoresis according to their
A. Size
B. Charge
C. Sequence
D. Orientation
13
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Concept Check:
DNA fragments move through the gel during
electrophoresis according to their
A. Size
B. Charge
C. Sequence
D. Orientation
14
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Methods for Analysis of DNA
• Nucleic acid hybridization and probes
• Single-stranded DNA can unite with other single-stranded
DNA or RNA, and RNA can unite with other RNA –
hybridization
• Foundation for gene probes – short DNA fragments of a
known sequence that will base-pair with a stretch of DNA
with a complementary sequence, if one exists in the
sample
• Useful in detecting specific nucleotide sequences in
unknown samples
– Southern blot method – DNA fragments are
separated by electrophoresis, denatured, and then
incubated with DNA probes. Probes will attach to a
complementary segment if present.
– Isolate fragments from a mix of fragments and find
15
specific gene sequences
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Methods for Analysis of DNA
• Fluorescent in situ hybridization (FISH) - probes are
applied to intact cells and observed for the presence
and location of specific sequences
– Useful for diagnostics or locating genes on a
chromosome
16
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Methods Used to Size,
Synthesize, and Sequence DNA
• DNA sequencing – determining the actual order
and type of bases for all types of DNA
• Most common sequencing technique is Sanger
technique
– Test strands are denatured to serve as a template to
synthesize complementary strands
– Fragments are divided into tubes that contain primers,
DNA polymerase, all 4 nucleotides, and fluorescent
labeled dideoxynucleotide
17
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Sanger Sequencing Method
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18
Methods Used to Size,
Synthesize, and Sequence DNA
• Studying the genome of an organism has spawned new
fields in biology:
– Genomics - the systematic study of an organism’s
genes and their functions
– Proteomics - the study of an organism’s complement
of proteins and functions mediated by the proteins
– Metagenomics - the study of all the genomes in a
particular ecological niche, as opposed to individual
genomes from single species
– Metabolomics - the study of the complete
complement of small chemicals present in a cell at
any given time
19
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Methods Used to Size,
Synthesize, and Sequence DNA
• Polymerase Chain Reaction (PCR) – method
to amplify DNA; rapidly increases the amount of
DNA in a sample
– Primers of known sequence are added, to indicate
where amplification will begin, along with special heat
tolerant DNA polymerase and nucleotides
– Repetitively cycled through denaturation, priming, and
extension
– Each subsequent cycle doubles the number of copies
for analysis
– Essentially important in gene mapping, the study of
genetic defects and cancer, forensics, taxonomy, and
evolutionary studies
20
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Polymerase Chain Reaction
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(a)
In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high
temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.
Cycle 1
5′
3′
5′
DNA Sample
3′
Segment of DNA
to be amplified
Heat to 948C
Denaturation
3′
5′
Strands
separate.
5′
3′
Priming
3′
Oligonucleotide
primers attach at
ends of strands to
promote replication
of amplicons.
508C to 658C
5′
3′
Amplicons Primer
Extension
Cycle
1
Primer
3′
5′
5′
728C
3′
Heat-stable DNA
polymerase synthesizes
complementary
strand.
5′
5′
3′
5′
1* fragment
(b) A view of the process after 6 cycles,
with 64 copies of amplified DNA.
Continuing this process for 20 to
40 cycles can produce millions of
identical DNA molecules.
3′
Polymerase
5′
3′
5′
2 copies
4 copies
8 copies
16 copies
32 copies
64 copies
3′
Cycle 2
Completed copies
Denaturation
New strand
Original
strands
New strand
Heat
to
948C
Priming
508C–658C
Cycle
2
Extension
728C
4 copies
Cycles 3, 4, . . . repeat same steps, with the copies of DNA
21
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Methods in Recombinant DNA
Technology
• Recombinant DNA technology – the intentional
removal of genetic material from one organism
and combining it with that of a different organism
– Objective of recombinant technology is cloning which
requires that the desired donor gene be selected,
excised by restriction endonucleases, and isolated
– The gene is inserted into a vector (plasmid, virus) that
will insert the DNA into a cloning host
– Cloning host is usually bacterium or yeast that can
replicate the gene and translate it into a protein
product
22
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Recombinant DNA Technique
23
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Characteristics of Cloning Vectors
• Must be capable of carrying a significant piece of
donor DNA
• Must be readily accepted by the cloning host
• Plasmids – small, well characterized, easy to
manipulate and can be transferred into appropriate
host cells through transformation
• Bacteriophages – have the natural ability to inject
their DNA into bacterial hosts through transduction
24
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Vector Considerations
• Origin of replication
is needed so it will
be replicated
• Vector must accept
DNA of the desired
size
• Gene which confers
drug resistance to
their cloning host
25
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Desirable Features in a Cloning Host
26
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Construction of a Recombinant,
Insertion, and Genetic Expression
• Prepare the isolated genes for splicing into a vector by
digesting the gene and the plasmid with the same
restriction endonuclease enzymes creating complementary
sticky ends on both the vector and insert DNA.
27
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Construction of a Recombinant,
Insertion, and Genetic Expression
• The gene and plasmid are placed together, their free
ends base-pair, and ligase joins them
• The gene and plasmid combination is a recombination
28
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Construction of a Recombinant,
Insertion, and Genetic Expression
• The recombinant is introduced into a cloning host
• The cloning host transcribes the gene and translates the
protein
29
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Screening for Recombinant
Microbes
• Use selective media to quickly identify recombinants
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Culture of cloning
host after incubation with
recombinant plasmid
Bacteria with
recombinant
plasmid
Bacteria lack
plasmid
(1)
(2)
Bacteria carrying
plasmid
Ampicillinresistance gene
Regular nonselective
medium with two
types of colonies
Selective medium with
ampicillin
Pure culture of
bacteria
containing
cloned gene
30
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Concept Check:
Which of the following is a primary participant in cloning an
isolated gene?
A. Restriction Endonuclease
B. Host Organism
C. Vector
D. Ligase
E. All of the above
31
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Concept Check:
Which of the following is a primary participant in cloning an
isolated gene?
A. Restriction Endonuclease
B. Host Organism
C. Vector
D. Ligase
E. All of the above
32
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Biochemical Products of
Recombinant DNA Technology
• Enables large scale manufacturing of lifesaving hormones, enzymes, vaccines
–
–
–
–
–
Insulin for diabetes
Human growth hormone for dwarfism
Erythropoietin for anemia
Factor VIII for hemophilia
HBV vaccine
33
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Recombinant Technology Examples
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34
Genetically Modified Organisms
(GMO, transgenic)
• Recombinant microbes
– Pseudomonas syringae – prevents ice crystals
– Bacillus thuringienisis – encodes an insecticide
• Many enzymes, hormones, and antibodies used in
drug therapy are manufactured using mammalian
cell culture
– Cell cultures can modify the proteins
• Microbes to bioremediate disturbed environments
• Oncolytic adenoviruses – host range consists of
cells that produce cancer-specific proteins
35
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Transgenic Plants
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Bacterium with
selected gene Chromosome
– Agrobacterium
tumefaciens: a natural
tumor-producing
bacterium
• Ti plasmid inserts
into the genomes of
the infected plant
cells
Isolated
gene for
herbicide
resistance
(a) The large plasmid (Ti) of
this bacterium can be
Chromosome
used as a cloning vector
Ti plasmid
for foreign genes that
code for herbicide or
T- DNA
disease resistance.
Agrobacterium cell
Gene splicaed
into Ti plasmid
Recombirium
Agrobacterium
Plant cell
(b) The recombinant plasmids
are taken up by the
Agrobacterium cells, wihich
multiply and copy the
foreign gene.
Agrobacterium with
Ti plasmid vector
(c) Genetically engineered
Agrobacterium is inoculated
into a culture of target plant
cells and infects the cells.
Process
in plant
(d) Bacterium fuses with the
plant cell wall and the Ti
plasmid enters . The T-DNA
carries the herbicide gene
into the plant chromosome.
Mature plants can be grown
from single cells, and these
transgenic plants will
express the new gene.
(e) Because the gene will be
part of the plant’s genome, it
will be transmitted to
offspring in seeds.
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36
Examples of Transgenic Plants
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37
Transgenic Animals
• Use a virus to transfect a
fertilized egg or early
embryo
• Transgenic animals will
transcribe and translate
eukaryotic genes
• Transgenic animals will
transcribe and translate
eukaryotic genes
38
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Transgenic Animals as
Disease Models
• Animal models have been designed to study
human genetic diseases
– Mouse models for CF, Alzheimer’s, sickle cell anemia
– Sheep or goats manufacture proteins and excrete them
39
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Examples of Transgenic Animals
40
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Genetic Treatments: Introducing DNA into the Body
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• Gene therapy: correct
or repair a faulty gene in
humans
– Ex vivo therapy:
normal gene cloned
in vectors, tissue
removed from the
patient
– In vivo therapy:
naked DNA or vector
is directly introduced
into the patient’s
tissues
1
2
1 Normal gene is isolated from
(1)
healthy subject.
2 Gene is cloned.
3 Gene is inserted into retrovirus vecto
4 Bone marrow sample is taken from
patient with genetic defect.
3
5 Stem cells from marrow are infected
with retrovirus, (enlarged view)
6 Transfected cells (red dots) are infused
into patient.
7 Patient is observed for
expression of normal gene.
7
5
4
6
Marrow stem cell
41
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Genome Analysis
• DNA Fingerprinting – Every individual has a unique
sequence of DNA
• Methods used include restriction endonucleases,
electrophoresis, hybridization, and Southern blot
42
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Copyright © McGraw-Hill Education. Permission required for reproduction or display.
4
Lanes
Probes for specific sequences added
4
4
3
3
2
2
1
1
3
2
Position of Migration
(b) Electrophoresis of DNA fragments separates them by size, with larger
fragments nearer the wells. Although invisible to the unaided eye at this
point, each lane contains a number of individual bands. Specific DNA
sequences are identified by hybridization with fluorescently labeled
nucleic acid probes or radioactive probes.
1
Samples
Visualized Bands
DNA
1 2 3 4 5 6 7 8 9
(a) Cells from different
samples are
processed to isolate
their DNA. The DNA
samples are exposed
to endonucleases
which snip them at
specific sites into a
series of different
fragments.
FORENSICTEST
Marker
PST Control
Suspect 2
Suspect 1
Marker
Evidence 2
Evidence 1
Victim
Marker
© DR. Michael Baird
(c) An actual DNA fingerprint used in a rape trial. Control lanes with known markers are in
lanes 1, 5, 8, and 9. The second lane contains a sample of DNA from the victim’ blood.
Evidence samples 1 and 2 (lanes 3 and 4) contain semen samples taken from the victim.
Suspects 1 and 2 (lanes 6 and 7) were tested Can you tell by comparing evidence and
suspect lanes which individual committed the rape?
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43
Genome Analysis
• Types of analysis
– SNP – single nucleotide polymorphism
– Markers
• VNTRs
• Microsatellite polymorphisms
44
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Genome Analysis
• DNA Fingerprinting is used to
•
•
•
•
Identify hereditary relationships
Study inheritance of patterns of diseases
Study human evolution
Identify criminals or victims of disaster
• Analysis of mitochondrial DNA is used to
trace evolutionary origins
45
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Genome Analysis
• Microarray analysis –
track the expression of
thousands of genes;
used to identify and
devise treatments for
diseases based on the
genetic profile of the
disease
46
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Concept Check:
If you wanted to identify which person licked a stamp, the
best method to use would be
A. Microarray Analysis
B. DNA Fingerprinting
C. Gene Therapy
D. Anti-sense RNA Gene Silencing
E. Any of the above would work
47
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Concept Check:
If you wanted to identify which person licked a stamp, the
best method to use would be
A. Microarray Analysis
B. DNA Fingerprinting
C. Gene Therapy
D. Anti-sense RNA Gene Silencing
E. Any of the above would work
48
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