Download Genetic Engineering

4th lesson
Medical students
Medical Biology
Genetic Engineering
Genetic Engineering
There are several terms that can be used to describe the technology, including
gene
manipulation,
gene
cloning,
recombinant
DNA
technology,
genetic
modification, and the new genetics. There are also legal definitions used in
administering regulatory mechanisms in countries where genetic engineering is
practised. The discovery of the structure of DNA by James Watson and Francis Crick
in 1953 provided the stimulus for the development of genetics at the molecular level,
and the next few years saw a period of intense activity and excitement as the main
features of the gene and its expression were determined. This work culminated with
the establishment of the complete genetic code in 1966 -- the stage was now set for
the appearance of the new genetics.
Gene expression
The flow of genetic information is from DNA to protein. Whilst a detailed
knowledge of gene expression is not required in order to understand the principles of
genetic engineering, it is useful to be familiar with the main features of transcription
and translation and to have some knowledge of how gene expression is controlled.
DNA cloning
One approach is to isolate the gene(s) responsible for the expression of a
protein or the formation of a product. The solution to this dilemma is to place a
relatively short fragment of a genome, which might contain the gene or other
sequence of interest, in an autonomously replicating piece of DNA, known as a
vector, forming recombinant DNA, which can be replicated independently of the
original genome, and normally in another host species altogether. These plasmids are
several types like F-plasmid (fertility) and R-plasmid (resistant). Propagation of the
host organism containing the recombinant DNA forms a set of genetically identical
organisms, or a clone. This process is hence known as DNA cloning. The initial
isolation and analysis of DNA fragments is almost always carried out using the
bacterium E. coli as the host organism, although the yeast Saccharomyces cerevisiae
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is being used to manipulate very large fragments of the human genome. S. cerevisiae
as yeast artificial chromosomes . Plasmid and phage vectors have been used to
express genes in a range of bacteria other than E. coli, and some phages may be used
to incorporate DNA into the host genome, for example phage λ . In the case of
plasmid vectors in E. coli, the most common situation,
the process may be divided into the following steps:
● Isolation of plasmid DNA containing the cloned sequence of interest.
● Digestion (cutting) of the plasmid into discrete fragments with restriction
endonucleases.
● Separation of the fragments by agarose gel electrophoresis.
● Purification of the desired target fragment .
● Ligation (joining) of the fragment into a new plasmid vector, to form a new
recombinant molecule .
● Transfer of the ligated plasmid into an E. coli strain (transformation) .
● Selection of transformed bacteria .
● Analysis of recombinant plasmids.
Restriction enzymes – cutting DNA
The restriction enzymes, which cut DNA at defined sites, represent one of the
most important groups of enzymes for the manipulation of DNA. These enzymes are
found in bacterial cells, where they function as part of a protective mechanism called
the restriction-modification system. In this system the restriction enzyme hydrolyses
any exogenous DNA that appears in the cell. To prevent the enzyme acting on the
host cell DNA, the modification enzyme of the system (a methylase) modifies the host
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DNA by methylation of particular bases in the restriction enzyme’s recognition
sequence, which prevents the enzyme from cutting the DNA. Restriction enzymes are
of three types (I, II, or III). Most of the enzymes commonly used today are type II
enzymes, which have the simplest mode of action. These enzymes are that cut at an
internal position in a DNA strand (as opposed to beginning degradation at one end)
they are known as endonucleases. Thus, the correct designation of such enzymes is
that they are type II restriction endonucleases, although they are often simply called
restriction enzymes. In essence they may be thought of as molecular scissors.
DNA ligation
To insert a target DNA fragment into a vector, a method for the covalent
joining of DNA molecules is essential. DNA ligase enzymes perform just this
function; they will repair (ligate) a break in one strand of a dsDNA molecule
provided the 5´ end has a phosphate group.
Recombinant DNA
We can now envisage an experiment in which a DNA fragment containing a
molecules gene (X) of interest (the target DNA) is inserted into a plasmid vector . The
target DNA may be a single fragment isolated from an agarose gel , or a mixture of
many fragments from, for example, genomic DNA . If the target has been prepared by
digestion with EcoRI, then the fragment can be ligated with vector DNA cut with the
same enzyme . In practice, the vector should have only one site for cleavage with the
relevant enzyme, since otherwise, the correct product could only be formed by the
ligation of three or more fragments, which would be very inefficient. There are many
possible products from this ligation reaction, and the outcome will depend on the
relative concentrations of the fragments as well as the conditions, but the products of
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interest will be circular molecules with the target fragment inserted at the EcoRI site
of the vector molecule (with either orientation), to form a recombinant molecule.
Transformation
The components of the mixture of recombinant and other plasmid molecules
formed by ligation must now be isolated from one another and replicated (cloned) by
transfer into a host organism. By far the most common hosts for simple cloning
experiments are strains of E. coli which have specific genetic properties. One obvious
requirement, for example, is that they must not express a restriction modification
system.
Selection
If all the competent cells present in a transformation reaction were allowed to
grow on an agar plate, then many thousands or millions of colonies would result.
Furthermore, transformation is an inefficient process; most of the resultant colonies
would not contain a plasmid molecule and it would not be obvious which did. A
method for the selection of clones containing a plasmid is required. This is almost
always provided by the presence of an antibiotic resistance gene on the plasmid
vector.
Gel analysis
Tracks corresponding to the vector plasmid and to recombinants are indicated.
The larger size of the recombinant plasmid is seen by comparing the undigested
plasmid samples, containing supercoiled and nicked bands, and the excision of the
insert from the recombinant is seen in the EcoRI digest.
Example of DNA cloning: pUC57= cloning vector carried required gene I(NS1B),
pET32= expression vector, HindII= restriction endonuclease , E. coli=Escherichia
coli .
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Transduction:
Some phage progeny released from the lytic may contain host DNA (transducing
phage) which is transferred into a new host in the next infection cycle. The foreign
host DNA can integrate by homologous recombination the process is called
Generalized Transduction .
The prophage may be exits from the bacterial chromosomes carrying small segment
of host genes with it and enter the lytic cycle transducing phages infect a new host
cells where recombination (crossing over) occur, the process is called Specialized
Transduction.
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Figure: Generalized transduction.
Figure: Specialized transduction: lac+ gene from P1 lac+ phage is inserted into
lac- bacterium by recombination. The resulting bacterium lac+.
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Protein engineering
One of the most exciting applications of gene manipulation lies in the field of
protein engineering. This involves altering the structure of proteins via alterations to
the gene sequence and has become possible because of the availability of a range of
techniques, as well as a deeper understanding of the structural and functional
characteristics of proteins. This has enabled workers to pinpoint the essential amino
acid residues in a protein sequence; thus, alterations can be carried out at these
positions and their effects studied. The desired effect might be alteration of the
catalytic activity of an enzyme by modification of the residues around the active site,
an improvement in the nutritional status of a storage protein, or an improvement in the
stability of a protein used in industry or medicine. Proteins that have been engineered
by the incorporation of mutational changes have become known as muteins.
The techniques involved in protein engineering are essentially based on
recombinant DNA technology and involve:
» various types of mutagenesis (to cause changes in specific locations or regions of a
gene to produce a new gene product).
» expression of the altered gene to form a stable protein.
» characterization of the structure and function of the protein produced.
» selection of new gene locations or regions to modify for further improvement as a
result of this characterization.
The commercial implications of the technical developments listed above are that a
large number of proteins, existing only in tiny quantities in nature, can now be
produced on. Furthermore, the yields of biochemical production can be increased
much faster than what was originally possible with classical fermentation.
Medical and forensic applications of gene manipulation
The diagnosis and treatment of human disease is one area in which genetic
manipulation is beginning to have a considerable effect. As many therapeutic proteins
are now made by recombinant DNA (rDNA) methods, and the number available is
increasing steadily. Thus, the treatment of conditions by recombinant derived
products is already well established. The techniques of gene manipulation impact
more directly on medical diagnosis and treatment, and use of rDNA technology in
forensic science. Recent progress in both of these areas is of course closely linked to
our increasing knowledge of the human genome, and new developments in medical
and forensic.
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