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DNA TECHNOLOGY
DNA Cloning
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Gene Cloning: Producing multiple
identical copies of a gene.
Recombinant DNA: Genes or DNA
from two difference sources are
combined into one molecule.
Genetic Engineering: Methods to
create recombinant DNA.
Plasmids as Vectors
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Plasmids: self replicating circular units of
DNA that can be used to carry cloned
genes.
Cloning Vector: DNA molecule that can
carry foreign DNA.
• Plasmids are a type of cloning vector.
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Restriction enzymes are used to cut open
the plasmid.
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The same restriction enzyme is used to cut the gene
out of its original source.
Complimentary sticky ends allows the sticky ends to
hydrogen bond together.
Ligase is used to reseal the phosphodiester bonds of
the DNA backbone.
Transforming Bacteria with the
Cloning Vector
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Transformation: the uptake of DNA
from the environment.
• Plasmids containing the gene of interest
can be introduced into bacteria which
then multiply and produce clones that
also carry the gene.
• These clones a can produce the gene
product in large quantities.
Inserting the Vector
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Plasmids that carry antibiotic
resistant genes are used.
• Bacteria that acquire the vector can
grow on growth medium containing
antibiotics where other bacteria cannot.
• This allows selection for bacteria only
carrying the vector.
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The gene of interest is inserted in the
middle of another gene and disrupts
its function.
Screening for the Bacteria
Containing the Cloned Gene
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In this particular example the bacteria
used are lacZ-.
• This means they are unable to metabolize
lactose because they have a mutated lacZ
gene.
• When they acquire the vector they can now
break down lactose.
• X-gal is a supplement that creates a blue
colored product when lactose is broken down.
• The gene of interest is inserted into the lacZ
gene and renders it inactive.
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When the insert is placed in the vector some of the
vectors take up the insert and others do not.
Screening for the Bacteria
Containing the Cloned Gene
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How do you distinguish between the
bacteria that have the insert and ones that
don’t?
• The bacteria are plated out on a medium
containing X-gal.
• Bacteria that have an intact lacZ gene will
hydrolyze X-gal and produce a blue product.
• Bacteria that contain the insert have a
disrupted lacZ gene and cannot break down Xgal so the colonies remain white.
The Polymerase Chain Reaction
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A process that produces many
identical copies of the same gene.
• Allows multiple copies to be produced
when there is very little DNA available.
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DNA from fossils
DNA from a crime scene
• The Process involves a DNA primer.
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Remember DNA polymerase can only
nucleotides to a preexisting DNA molecule.
The primer is homologous to the gene of
interest and extends the DNA
complimentary to the gene.
Polymerase Chain Reaction
DNA Analysis
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What if we wanted to know the
sequence of gene?
• To compare allele differences associated
with heredity disorders.
• To compare closely related two species
are.
• To determine whether or not some one
committed a crime.
Restriction Fragment Analysis
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Uses electrophoresis to separate DNA
fragments after they have been digested
with restriction enzymes.
• Mutated forms of an allele are going to exhibit
a different band pattern on the gel.
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This is because the restriction sites are in different
places on the allele yielding fragments of DNA that
vary from the bands of the non mutated allele.
Scientists can match band patterns on the gel but
only know the relative sizes of the DNA fragments.
They do not know the actual DNA sequence.
Electrophoresis
The Gel
The Southern Blot
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This procedure allows the identification of specific
sequences of DNA.
• An imprint of the DNA is made on a filter that is placed
on top of the gel.
• The DNA on the filter is now “denatured” and made
single stranded.
• The filter is then incubated with a labeled probe
(radioactive) that is complimentary to the gene of
interest.
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Since the DNA on the filter is single stranded it will bind to
the complimentary sequences of the probe.
• The filter is washed to remove excess probe that is not
bound and the filter is then exposed to film.
• The film shows where the radioactive probe was
complimanty to the gene.
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This is known as a autoradiograph.
The Southern Blot
Restriction Fragment Length
Polymorphisms
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RFLP Markers: Adjacent to genes are
satellite sequences that do not code for
anything but vary between individuals.
• Because the restriction sites are in different
places within the RFLP sequences each person
has a DNA “fingerprint”.
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Even though the sequence of the gene itself is the
same from person to the next the RFLP sequences
account for different banding patterns.
DNA can indicate the presence of an individual at the
scene of a crime
The Autoradiograph
• http://www.dnalc.org/ddnalc/resources/pcr.h
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