Download polymerase chain reaction

There once was a small black-and white-striped fish (that might
even be in your aquarium) that was being studied by Dr. Mark
Keating of Harvard. And Dr. Mark asked the question, “if the
zebrafish can regenerate fins and eye parts, could it regenerate. . .
. ?”
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So he took this 1-inch long fish and cut away about 20% of
their two chambered hearts. The incisions through the
abdomen were blotted to stop bleeding, the fish were returned
to the water and 8 out of 10 survived the experiment. “They
sort of hang out at the bottom of the tank” it was reported.
But within 10 days, the fish began to swim normally and were
healthy as nonexperimental fish.
After 2 months, the fish had totally regenerated their hearts,
replacing all the tissue and the cells were vigorously beating
with little or no scarring of the tissue.
Why should we care??
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DNA TECHNOLOGY MAKES IT POSSIBLE TO
CLONE GENES FOR BASIC RESEARCH AND
COMMERCIAL APPLICATIONS
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How do you (or did you) use a bacterial plasmid to clone a
gene?
1) Use of restriction enzymes
2) Mix the cut plasmid and source of the gene
-jellyfish in the pGLO lab
-but what if it was a heart muscle repair gene?
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3) What occurs during the mixing of the cut plasmid and
the cut DNA of interest?
4) But how do you know that recombination has even
occurred?
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5) All you have at this point is, hopefully, plasmid with
a new gene inserted.
6) Introduce this modified plasmid into bacteria.
7) How will we select for the recombinant bacteria?
That is, how will you locate them? How will you
know which ones have taken up the plasmid and
which ones haven’t
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8) Ways to distinguish the recombined bacteria:
-antibiotic resistance
-reporter gene or marker such as GFP
-if you were isolated the heart muscle tissue
repair gene you could use GFP
-some kind of fluorescence
9) Select those bacteria that have been transformed
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10) But you have to have lots of the product of this gene so
you must let these bacteria reproduce.
11) So this zebrafish gene that is responsible for repairing
torn heart muscle tissue might be in humans or could be
implanted in human cardiac cells.
12) How do find out if we even already have this gene but we
are just not “using” it?
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13) By sequencing the gene of the zebrafish and comparing its
sequence to the database for our genome.
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What has this procedure contributed to genetics?
1) Study gene products
2) Produce necessary proteins: insulin, human growth
hormone, receptor proteins
3) Produce transgenic organisms: rice that will produce levels
of vitamin A for those people / areas deficient in vit. A
4) Study gene regulation or gene structure
5) Develop a gene library
6) Amplify a particular gene of interest
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Dideoxy Method of DNA Sequencing
1) A modified form of the normal deoxyribonucleotide
triphosphate (dNTP) is used.
2) The modified form is called a dideoxy- because its 3’
hydroxyl is removed which prevents elongation of the
DNA strand.
3) These dideoxy’s are also tagged with a particular molecule
that when exposed to a laser will be excited and emit a
different color.
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4) SS DNA of interest (lots of copies) is/are isolated and mixed
with:
-dNTPs
-DNA primer
-DNA polymerase
-Buffer
-fluorescent-tagged ddNTPs
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5) The gene of interest is then copied but whenever a ddNTP is
added to the growing strand, replication stops.
6) This will produce mixture of different sized fragments that are
complementary to the gene of interest.
7) The exact lengths can be used to determine the position of each
base in the growing chain.
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8) At this point you have fragments of ds DNA and the
fluorescently tagged bases are complementary to your gene.
9) Heat up the DNA, separate the strands; electrophorese the
different length strands (capillary electrophoresis)
10) As the fragments run off the gel, a laser excites the different tag
on the base and emits a different color for each base.
11) This color and therefore base is recorded.
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12) Remember, we have just determined the order of the bases in
the complementary strand so the actual gene is complementary
to the readout.
13) Dye Sequencing Animation from CSHL
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Another method to clone a gene is by the polymerase chain
reaction
What were the requirements needed to perform this technique?
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What was PCR’s contribution to genetics?
1) The ability to make millions of copies of a gene
2) Amplifies a very specific region: PCR Animation
3) Use in forensics
-let’s let the specific region being amplified be amplified
in different amounts in various individuals. A region could
be present as 1 “repeat” or 4 “repeats” or 40 “repeats.” So
the region is highly variable.
-and now let’s say we have 5 to 10 different regions where
various kinds of repeats could be located.
-the odds that two random individuals would share this
same genetic pattern by chance is about 1 in 10 billion.
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4) Its use in paternity diagnosis:
-same as in forensics
5) Its use in medical diagnosis:
-let the Alu insert be present whenever a severe disorder of a
disease occurs. This “Alu insert” would be a marker for a
particular disease. Statistics would determine the probably of
the development of the disease when the marker (s) is present
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6) Its use in evolutionary applications
-the more closely associated DNA sequences are between
organisms, the more closely related they are.
-the more specific markers, regions, genes, etc. are in
common, the more associated are the organisms.
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Figure 20.1 An overview of how bacterial plasmids are used to clone genes
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Figure 20.2 Using a restriction enzyme and DNA ligase to make recombinant DNA
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Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 3)
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Figure 20.4 Using a nucleic acid probe to identify a cloned gene
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Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene
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Figure 20.6 Genomic libraries
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Figure 20.7 The polymerase chain reaction (PCR)
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Figure 20.8 Gel electrophoresis of macromolecules
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Restriction Fragment Length Patterns
RFL Patterns can be used to distinguish different alleles

Alleles are different forms of a certain gene. They can differ because
of a different base sequence. This difference in base sequence then
allows one form of the allele to be functional, and the other not.

Let’s say we have 2 different cloned gene samples and that the base
sequence near these alleles has a restriction site or sequence. If in one
of the alleles there is a different base and thus changing the restriction
site, the restriction enzyme will not cut there.

This inability to cut at one of the sites will produce DNA fragments
that are of different sizes / numbers.

So when the fragments from allele 1 and the fragments from allele 2
are run on a gel, different bands are produced for each of the alleles.

This shows that there is a single base difference between the two
alleles of the two different DNA samples.
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Figure 20.9 Using restriction fragment patterns to distinguish DNA from different alleles
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Southern Blotting
What if we want to search for a gene of interest or a sequence of interest amongst
a collection of DNA fragments?

We could “see” which fragment amongst the 100’s has our gene of
interest (GOI) by attaching a radioactive probe to it. This is what a
Southern Blot does, it isolates a strand of DNA by hybridization with a
radioactive probe.

Purposes of Southern Blotting:
 Identifies all the fragments of DNA in your sample that have the
sequence of interest.
 Can be used to find sequences in noncoding sections (introns) and
therefore differences / similarities between organisms.
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Figure 20.10 Restriction fragment analysis by Southern blotting
Alkaline solution draws the DNA up
into the nitrocellulose paper and
denatures the DNA into single strands
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RFLPs
Differences in nucleotide sequences between alleles or in the introns can create
different sized fragments of DNA because these differences change the location
of where a restriction enzyme can cut.

These different sized fragments are called Restriction Fragment Length
Polymorphisms.

These RFLPs can serve as genetic markers telling you what your
“banding pattern” looks like as compared to someone else.

Look at step 5 of the next slide
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Figure 20.10 Restriction fragment analysis by Southern blotting
Alkaline solution draws the DNA up
into the nitrocellulose paper and
denatures the DNA into single strands
Individuals I and II have
the same genetic marker.
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Human Genome Project
It’s about:

determining the entire sequence of all 22 pairs of autosomes plus the X
and Y chromosome

determining the sequence of many other species: E. coli, S. cerevisiae
(yeast), Drosophila (fruit fly), Arabadopsis, mouse, C. elegans
(nematode)
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Entire Genomes Can Be Mapped
Genetic Mapping

The markers or RFLPs can be used to determine recombination
frequencies which will tell you how far apart they are.

These markers could also be genes

This helps put genes “in order.”
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Entire Genomes Can Be Mapped (cont’d)
The Ordering of DNA Fragments

You can do this by cutting up the whole chromosome with restriction
enzymes and putting the fragments together.

You will use restriction enzymes that create some overlapping amongst
the fragments

Chromosome walking: this helps to provide a series of markers along a
chromosome
 You first need to know something about a gene or marker and then
make a probe, probe 1, to bind to its 3’end.
 Take your DNA and cut it with two different restriction enzymes and
clone copies of these fragments.
 Expose your probe to Library II, the DNA of the cloned fragments cut
with a different restriction enzyme.
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Entire Genomes Can Be Mapped (cont’d)
The Ordering of DNA Fragments (cont’d)

Chromosome walking: this helps to provide a series of markers along a
chromosome
 Your probe 1 will bind to a fragment of the DNA from library II.
 Isolate this fragment
 Make a probe, probe 2, for its 3’ end.
 Expose probe 2 to the DNA from library 1 and this will bind further
along the DNA, hence walking down the DNA fragment.
 If you keep repeating this, you move all the way down the fragment
with these probes and where these probes bind serve as
sequences or markers in a known order.
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Figure 20.11 Chromosome walking
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Figure 20.12 Sequencing of DNA by the Sanger method (Layer 1)
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Figure 20.12 Sequencing of DNA by the Sanger method (Layer 2)
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Figure 20.12 Sequencing of DNA by the Sanger method (Layer 3)
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Figure 20.12 Sequencing of DNA by the Sanger method (Layer 4)
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Figure 20.13 Alternative strategies for sequencing an entire genome
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Table 20.1 Genome Sizes and Numbers of Genes
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Figure 20.14a DNA microarray assay for gene expression
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Figure 20.14b DNA microarray assay for gene expression
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Practical Applications of DNA Technology
1.
Diagnosis of Diseases
a)
Knowing the sequence of a genome such as that of HIV, you can
determine whether or not it is present in a blood/semen/tissue sample.
b) Genes for a variety of diseases have been cloned and therefore can be
identified: hemophilia, cystic fibrosis, Duchenne muscular dystrophy
c)
These genetic diseases can be detected by exposing the DNA to
radioactive probes that will bind to the mutant alleles.
d) RFLPs can be used to identify a marker that is associated with a genetic
disorder.
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Practical Applications of DNA Technology
2.
Human Gene Therapy
a)
Most effective if there is a single genetic disorder.
b) Can we replace the gene or at least insert a good copy of it?
c)
3.
Insert it into the somatic cells so then then the gene product can be
released into the blood stream.
Pharmaceutical Products
a)
Human insulin
b) Human growth hormone
c)
Tissue plasminogen activator: helps to dissolve blood clots and thus
reduces the risk of a blood clot after a heart attack.
d) Protein products that block receptors (HIV)
e)
Produce viral noninfectious protein products for vaccines
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Figure 20.16 One type of gene therapy procedure
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Practical Applications of DNA Technology
4.
Forensics
a)
The DNA sequence of each individual is unique and these variations
produce different RFLPs.
b) Compare blood from the suspect, the victim and the crime scene to
associate the suspect.
c)
Compare blood of mother, child and possible father.
d) STR’s or Simple Tandem Repeats: this is where a short series of
repeating bases can occur 10 to 100 times. PCR is used to selectively
amplify one of these regions and will run out differently on a gel as
different sized fragments distinguish you from me.
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Figure 20.17 DNA fingerprints from a murder case
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Practical Applications of DNA Technology
5.
Environmental Uses
a)
Microorganisms can extract toxic metals from soil
b) Bacteria can be engineered to degrade toxic organic compounds.
6.
Agricultural Uses
a)
Transgenic Animals: carry genes from other species
i.
How is a transgenic animal made: remove eggs; fertilize them in
vitro; insert the cloned, desired gene into the nuclei of the eggs;
some eggs will integrate the injected DNA into the genomic DNA;
gene product is expressed; these eggs are them implanted into
mother; embryo develops and it has the “new” genes.
ii.
Isolate a breed of cow that contains a gene that makes more muscle
mass and isolate the gene, clone it and insert it into other cows.
iii. Insert a gene for blood clotting into cows and have its gene product
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released into the milk of the cow. Purify it.
Practical Applications of DNA Technology
7.
Genetic Engineering in Plants
a)
To:
i.
Delay ripening
ii.
Provide frost resistance
iii. Provide pest resistance
iv. Resist spoilage
v.
Vitamin A or other vitamin deficiency correction
vi. Increase nitrogen fixation by plants
vii. Create drugs / proteins for use as medicines / vaccines.

Vaccine for hepatitis B

Tobacco plants are being used to create antibiotics against
bacteria that cause tooth decay.
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Figure 20.20 “Golden” rice contrasted with ordinary rice
Increase the nutritional value of food by inserting the gene to make
beta-carotene. Beta-carotene makes vitamin A in our bodies so this
transgenic rice could help prevent vit. A deficiencies. 70% of children
under the age of 5 in Southeast Asia suffer from Vit A deficiency.
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Practical Applications of DNA Technology
b)
How do you get these genes into plants?
i.
By the use of a plasmid called a Ti plasmid. This plasmid comes from
a bacterium that causes tumors in plants. The Ti plasmid will insert
some of its DNA into the plant’s DNA causing the tumor but
researchers work with a non-tumorigenic form.
ii.
So give the non-tumorigenic plasmid a new gene and then infect plant
cells in culture or directly into the plant cells. The plant cells then
insert the new gene into their genome.
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Figure 20.19 Using the Ti plasmid as a vector for genetic engineering in plants
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Genetically Modified Organisms or GMOs
A lot of concern surrounds recombinant microbes.
1.
Can new pathogens be created?
a)
What if a bacterium had cancer genes inserted into it?
b) What about a flu virus getting these cancer genes?
c)
2.
Is this possible? Is there a real risk?
Implemented Safety Measures
a)
Strict measures to prevent microbes from escaping the labs
b) Microbe strains are modified so they cannot survive outside the lab.
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Genetically Modified Organisms or GMOs (cont’d)
A GMO is any organism that has acquired one or more genes by artificial means.

No animals yet but plenty of crops

Biosafety Protocol of 2000: exporters must identify GMOs present in
their bulk food shipments and the importing countries can decide if there
is a health or environmental risk.

There is the fear that crops carrying genes from other species are a health
concern and could do ecological harm.
 So a transgenic plant could transfer the new genes to a closely related
species that was never intended to be modified. So if a weeds we want to
control picked up a gene from a modified relative that was herbicide
resistant, we would have a hard time controlling the weed.
 No scientific evidence that genetically modified crops to resist pests
pose a risk.
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Figure 20.x2 Injecting DNA
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The Technique of Restriction Fragment Length
Polymorphisms
1) What is a Riff-Lips (RFLP)? Mutations within a
genome, coding or noncoding regions, can affect where
restriction enzymes can cut. The addition or deletion of
a restriction site will then affect the size and number of
fragments.
2) This can occur with in a gene (coding region) or a region
flanking the gene (noncoding region)
3) This will produce different “restriction maps” between
individuals on the same chromosome.
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4) The technique
-obtain DNA
-cut with restriction enzymes
-electrophorese
-separation of size of fragments based on size
-stain with ethidium bromide
-photograph and analyze
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What has this technique contributed to genetics?
1) Genetic markers in families
2) Marker for diseases: sickle cell has only one amino acid
change because an AT base pair is changed to a TA base pair.
This changes the codon and valine is inserted instead of
glutamic acid in the Beta-globin polypeptide of hemoglobin.
This single base pair substitution creates a restriction site
thus creating a different RFLP pattern between affected and
normal individuals.
3) Phenylketouria (PKU) is diagnosed by a RFLP caused in a
flanking region of a gene responsible for making the
enzyme phenylalanine hydroxylase. Tested by amnio- or
chorionic villus sampling in the fetus.
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4) DNA fingerprinting
5) Mapping of DNA can be done by RFLP: if two RFLPs are
inherited frequently or if a RFLP marker and an allele are
frequently inherited, this can be used as a measure closeness
of the two segments of DNA (the two loci).
6) Evolutionary relationships between plants / animals.
7) Find mutations
8) Sequences of DNA can be determined even though you do
not know its function.
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