BI304-lecture_12[1].ppt - MiNDS

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Dec 14, 2012 (4 years and 7 months ago)

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Recombinant DNA and Genetic
Engineering
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1

Genetic Engineering
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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.

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.

Making Use of Natures Genetic Resources

In 1950’s it was discovered that bacteria contain
autonomously replicating extrachromosomal DNA
entities which were called plasmids. It was noted in
the early 1970’s that plasmids would be the ideal
vectors for developing recombinant DNA technology.
Plasmids are one of the major tools of genetic
engineering.

Plasmids

Vectors for Recombinant DNA Technology

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
.

Cloning Vectors are Tailored for
Particular Organisms

Restriction endonucleases

It was discovered in the 1960’s 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.

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
.

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.

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.

Examples of Restriction Enzymes

Example of a Generic Cloning Experiment

Sticky ends!

The General Process of Cloning a DNA
Fragment:

A)
Digest your target DNA with restriction enzyme.

B)
Digest your vector DNA with the
same restriction

enzyme.

C)

Ligate these fragments together.

D)
Transform your clones into E. coli.

E) Isolate large quantities of your DNA of interest.

Outline of the whole
cloning process

This could be “any” piece of
DNA

Grow bacteria and make
large quantities of your
recombinant plasmid.

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.

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.

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.

Directional cloning into the
Multiple Cloning Site (MCS)
of pUC19.

Direct identification of
relevant clones by
LacZ

inactivation using pUC19.

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.

Which Colonies Contain my Cloned
Fragment DNA?

Next lecture will focus on specific techniques such
as PCR,
in vitro

mutagenesis and DNA library
construction.