Transcript Ch 8 Genetic Technology and Diagnostics

Genetic
Engineering
1
Genetic Engineering: Enzymes for Dicing and
Splicing Nucleic Acids
•Restriction endonucleases
- enzymes capable of recognizing foreign DNA
and breaking the phosphodiester bonds
between adjacent nucleotides on both strands of
DNA
-
protects bacteria against incompatible DNA of
bacteriophages
-
allows biotechnologists to cleave DNA at
desired sites
-
necessary for recombinant DNA technology
-
recognize and clip at palindromes
Restriction Endonucleases
•Make staggered symmetrical cuts that leave short tails
called “sticky ends”
- cut four to five bases on the 3’ strand and on
the 5’ strand, leaving overhangs on each end
-
adhesive tails will base-pair with complementary
tails on other DNA fragments or plasmids
Restriction Endonucleases
-
restriction fragments:
pieces of DNA produced
by restriction
endonucleases
-
restriction fragment
length polymorphisms
(RFLPs): differences in
the cutting patterns of
specific restriction
endonucleases
Analysis of DNA
•Gel electrophoresis
- produces a readable pattern of DNA fragments
-
samples are placed in compartments in a soft agar
gel and subjected to an electrical current
-
phosphate groups have a negative charge, which
causes DNA to move toward the positive pole in
the gel
-
larger fragments migrate more slowly; smaller
fragments migrate more quickly
-
position of fragments are determined by staining
the gel
-
creates a genetic fingerprint
Revealing the Patterns with Electrophoresis
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Electrophoresis
Restriction endonucleases
selectively cleave sites
of DNA
Known
DNA size
markers
Samples
1
2
3
4
5
Wells
Restriction
fragments
DNA for sample 3
Larger
Smaller
1
2
3
4
(–)
5
Samples
Wells
Size markers
(b)
(+)
(a)
DNA migrates toward
positive electrode.
© Kathy Park Talaro
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Polymerase Chain Reaction:
A Molecular Xerox Machine
for DNA
•Rapidly increases the amount of
DNA in a sample without the need
for making cultures or
carrying
out complex purification
techniques
•Sensitive enough to detect cancer
from a single cell or diagnose an
infection from a single gene copy
•Rapid enough to replicate target
DNA from a few copies to billions of
copies in a few hours
Polymerase Chain Reaction
•Primers: DNA strands 15 – 30 bases
long that serve as landmarks where
DNA amplification should begin
•DNA polymerase from thermophilic
bacteria
- “Taq” polymerase isolated
from Thermus aquaticus
- remain active at elevated
temperatures used in PCR
•Thermal cycler: automatically
performs the cyclic temperature
changes required for PCR
Recombinant DNA Technology
•Remove genetic material from one organism and
combine it with that of a different organism
•Bacteria can be genetically engineered to mass produce
substances such as hormones, enzymes, and vaccines
difficult to synthesize by usual industrial methods
Recombinant DNA Technology
•Genetic clones/cloning
- involves removal of a selected gene from an
animal, plant, or microorganism and propagated
in a host microorganism
-
gene must be inserted into a vector (usually a
plasmid or a virus)
-
vector inserts the gene into the cloning host
-
cloning host is usually a bacterium or yeast
which can translate the gene into the desired
protein
Cloning Vectors
•Plasmids
- small, well characterized, easy to manipulate
- can be transferred into appropriate cells through
transformation
•Bacteriophages: have the natural ability to inject DNA
into bacterial hosts through transduction
•Vectors typically contain a gene that confers drug
resistance to their cloning host
- cells can be grown on drug containing media
-
only those cells that harbor a plasmid will be
selected for growth
Cloning vectors
• Carry a significant piece
of the donor DNA (gene
of interest)
• Readily accepted DNA
by the cloning host
• Contain an origin of
replication
• Contain a selective
antibiotic resistant gene
• Ex. Plasmids, phages
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Producing Recombinant DNA is easy
• Start with a cloning vector (special plasmid) and DNA with your gene
of choice.
• Cut the cloning vector and your desired gene out of the parent
chromosome with specific enzymes.
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Producing Recombinant DNA is easy
• Mix the vector and the gene together with a ligase enzyme which
“seals” the DNA together.
• Use various techniques to insert the vector +gene into a new cell.
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Producing Recombinant DNA is easy
• Grow cell on selective or
differential media to find out which
cells possess the recombinant
plasmid.
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Modified bacteria
• Pseudomonas syringae
– Natural bacteria that grow on plants but promote frost crystals
– Alteration of the normal frost gene now prevents frost crystals
from forming on plants (applied with crop duster to compete with
the natural bacteria in the field)
• Pseudomonas fluorescens
– This bacteria was engineered to contain an insecticide gene. The
bacteria is sprayed on fields with crop dusting planes. The bacteria
grow on the plants and when the insects start to eat the plant
they will also eat some bacteria with the insecticide. The
ingestion of insecticide kills the insects.
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Transgenic plants using bacteria
• Agrobacterium tumefaciens
– Ti plasmid contains gene of interest, and is
integrated into plant chromosome
– Ex. tobacco, garden pea, rice
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Schematic of Agrobacterium tumefaciens transferring and integrating
the Ti plasmid into the plant chromosome.
Fig. 10.11 Bioengineering
of plants
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Transgenic animals
• Pharmaceutical production
• Knockout mouse
– Tailor-made genetic defects
•
•
•
•
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Cystic fibrosis
Gaucher’s disease
Alzheimer’s disease
Sickle-cell anemia
Allow us to research cures to these diseases
24
Nkx2.2 KO
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Genotypic Diagnostics:
DNA Analysis Using Genetic Probes
•Hybridization
- used to identify bacterial species by
analyzing the sequences of nitrogenous
bases in DNA
-
probes: small fragments of single-stranded
DNA or RNA complementary to the specific
DNA sequence of a particular microbe
-
unknown test DNA is extracted from
cells and bound to blotter paper
-
probes are added to the blotter paper
and visible signs that probes have been
hybridized to the test DNA
DNA Analysis Using Genetic Probes
•Probes can be labeled with chemically luminescent materials that can
be measured by “light meters,” which avoids the use of radioactivity
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ATGGTC
Addition
of probes
Blotter paper
containing DNA
from unknown
organism
CTGGTA
No complementary
base pairing
No color
development
on the paper
Complementary base pairing
TACCAG
ATGGTC
Color development
on the paper
Nucleic Acid Sequencing
and rRNA Analysis
•One of the most viable indicators of
evolutionary relatedness and affiliation is the
comparison of 16s rRNA sequences
- 16s rRNA is part of the 30s subunit of
the bacterial ribosome
-
16s rRNA is highly conserved across
species and evolutionary time
-
perfectly suited for bacterial identification
and diagnosis of infection
rRNA Analysis
•Fluorescent in situ hybridization (FISH)
- rapidly identifies 16s RNA sequences
without first culturing the organism
-
relies on dyes to emit visible light in response to
UV radiation
-
turnaround time for identifying suspect
pathogens present in blood cultures has been
reduced from 24 hours to 90 minutes
Peptide Nucleic Acid FISH Testing for S. aureus
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S. aureus
PNA probe
Nucleic acid
Peptide
Fluorescent
molecule
90 minutes
Gram-positive
cocci in clusters
S. aureus
on a slide
Positive blood
culture tube
rRNA in
ribosome
Image provided by AdvanDx, Inc.