Recombinant DNA and Genetic Engineering-1

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Recombinant DNA and Genetic Engineering-1
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Genetic Engineering- Tools of the Trade
Throughout science there are examples where
technical developments lead to quantum leaps in
our knowledge, understanding and experimental
capabilities. This is the case with recombinant
DNA technology/genetic engineering. Main tools
of the trade are plasmids (and other vectors)
and restriction endonucleases.
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Genetic Engineering Viewed as a Whole Process
It is now routine to be able to identify a gene
or fraction of a gene that we are interested in
and isolate this DNA in a test tube. Our DNA of
interest can then be coupled to a plasmid (or
other vector) in a way that we can dictate. We
can then put this cloned DNA inside a bacterium
which we can then grow up in relatively vast
quantities to obtain large amounts of our target
DNA. We can now further alter this DNA or harvest
its protein products.
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Making Use of Natures Genetic Resources
Plasmids
In 1950s it was discovered that bacteria contain
autonomously replicating extrachromosomal DNA
entities which were called plasmids. It was noted
in the early 1970s that plasmids would be the
ideal vectors for developing recombinant DNA
technology. Plasmids are one of the major tools
of genetic engineering.
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Vectors for Recombinant DNA Technology
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Different Vectors for Different Species
Because different species use different promoter
elements and other regulatory features, it is
important to use a vector that is specific for
the type of organism you are working with (see
next table!). Example, E. coli plasmids will not
replicate and function in yeast! Shuttle vector
designed to have features required to function in
two or more species. Virtually all yeast plasmids
are engineered to function in E. coli.
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Cloning Vectors are Tailored for Particular
Organisms
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Restriction endonucleases
It was discovered in the 1960s that bacteria
possess enzymes (now called restriction
endonucleases) that have the ability to digest
foreign species of DNA. This allowed bacteria to
protect themselves from invading viruses. To
protect their own DNA bacteria would methylate
specific sequences that would render them
immune to their own destructive enzymes.
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The original cloning concept and proof of
principle involved a) Realising the possibility
of using a restriction enzyme to cut and paste
DNA. b) Experiment carried out by Chang and
Cohen circa 1974 showing that DNA from
Staphylococcus aureus can be cloned into an E.
coli plasmid and replicated in E. coli.
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Properties of Restriction Endonucleases
Different than general endonuclease in that they
recognise and cut at specific sequences. These
sequences are normally around 4-6 base pairs long
but can be as much as 10 base pairs. Most
restriction enzymes make two single-strand
breaks, one in each strand thus generating two
3-OH and two 5-P base pair overhanging termini.
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Depending on the restriction enzyme the cuts
generated in the DNA are described as producing
cohesive (or sticky) ends, or blunt ends. In
practice it is much easier to ligate together two
sticky ended DNA fragments than two blunt
fragments.
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Examples of Restriction Enzymes
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Example of a Generic Cloning Experiment
Sticky ends!
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The General Process of Cloning a DNA Fragment

• Digest your target DNA with restriction enzyme.
• Digest your vector DNA with the same restriction
  enzyme.
• Ligate these fragments together.
• Transform your clones into E. coli.
• E) Isolate large quantities of your DNA of
  interest.

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Outline of the whole cloning process
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Which Bacteria Contain my Fragment of Interest?
During the ligation reaction, not all plasmid
vectors will have foreign DNA fragments ligated
into them. Some digested vector will religate to
itself. These products are useless and need to be
removed from any further analysis. Systems to
identify relevant clones have been developed. Two
common methods employed and work well together.
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1. Directional Versus Bidirectional Cloning
The use of two restriction enzymes allows a
drastic reduction in the religation of vector
DNA. This enhances the chances of retrieving
clones that you want.
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2. Inactivation of a Reporter Gene
Plasmids have been designed that carry the E.coli
LacZ gene. When grown on medium containing the
substance X-Gal, these strains are blue. If the
gene is inactivated then the colonies are white.
By cloning DNA into the middle of the LacZ gene
it will be inactivated and then can be used as a
reported for presence of cloned DNA fragments.
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Direct identification of relevant clones by LacZ
inactivation using pUC19.
Directional cloning into the Multiple Cloning
Site (MCS) of pUC19.
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Clone Identification by Nucleic Acid Hybridisation
Sometimes it is impractical to use directional
cloning or inactivation of a reporter gene. This
could be due to availability of good restriction
sites in your DNA of interest or just to lack of
a good vector to use. In these cases Southern
blotting can be used to identify relevant clones.
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Which Colonies Contain my Cloned Fragment DNA?
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Next lecture will focus on specific techniques
such as PCR, in vitro mutagenesis and DNA library
construction.