GENETIC ENGINEERING

(Recombinant DNA Technology)

Genetic Engineering - the manipulation of genes using technology. It involves cutting DNA into
recognizable pieces and rearranging these or combining two different genomes into one.

Recombinant DNA technology has its basis in certain enzymes called Restriction
Endonucleases. These are enzymes that cut DNA in precise locations. They are present in
bacteria and function in protecting the bacteria from intruding viral DNA.

They work by cutting the foreign DNA via a process called restriction. The enzymes restrict the
replication of the viral DNA by cutting it into fragments. These endonucleases recognize specific
nucleotide sequences (restriction sites) in DNA and cut at a specific place within the recognition
sequence. DNA cut by restriction enzymes are called restriction fragments.

Restriction enzymes’ splicing of DNA may result in single stranded ends (‘sticky ends’) or blunt
ends on the restriction fragments. Sticky ends will H- bond with complimentary single stranded
ends of DNA cut with the same enzyme.

This property of restriction enzymes allows DNA from different sources to be cut and joined
later resulting in a completely new genome in the organism.

STEPS IN RECOMBINANT DNA TECHNOLOGY

1. A copy of the gene of interest must be obtained.

2. The gene must be placed into a vector.
3. The vector is then placed into the host cell.
4. Transformed hosts are identified and separated.
5. The gene is cloned.
Isolating the gene of interest

The oldest method is the ‘shot-gun’ method. Chromosomes containing the gene
are combined with restriction enzymes and are cut up by these. This method is
‘hit and miss’ since there is no guarantee that the complete gene that is required
will be on any of the fragments.
Another way to get a copy of the gene desired is to use Reverse Transcriptase to
synthesize cDNA from mRNA extracted from the cell. DNA Pol 111 is then used to
synthesize another strand to make the cDNA double stranded.

A third method to obtain the gene of interest is by artificial synthesis. Once the
gene sequence is known, nucleotides can be synthesized and joined in the lab to
make the gene.

Placing the gene into the vector

A vector is the means used to introduce foreign DNA into the host cell. Two
vectors are used: 1) bacteriophage - a virus that infects bacteria and 2) plasmid-
small, circular, non-chromosomal DNA found in bacteria.

Plasmids are removed from bacteria by treating them with calcium chloride. This
creates holes in the cell membrane, essentially breaking open the bacteria.

The plasmid or viral DNA is cut with a restriction enzyme. The gene of interest
would also be cut with the same restriction enzyme.

The vector DNA is then combined with the gene of interest and DNA ligase is used
to join the two together. The two different DNA combined into one genome is
called Recombinant DNA (rDNA).

Placing the vector into the host

The host is the organism that will facilitate the replication or cloning of the gene
of interest. Hosts are usually bacteria. The most common host used is Escherichia
coli. E. coli multiplies rapidly and is easy to manipulate.

The rDNA is inserted into the bacterial host by punching small, temporary holes in
its membrane. The holes are created by treating with CaCl2 and heating or
delivering a brief electrical pulse. More recently, DNA guns and micro needles
have been used to insert rDNA into hosts.

If the vector is a phage, then the hosts are allowed to be infected by the virus.
Bacteria that take up the rDNA are described as being transformed.

Selecting transformed hosts

To determine which bacteria have been transformed, the culture is grown on a

medium with an antibiotic for which the plasmid used as vector has the resistant
gene. As such, only those bacteria that had taken up the vector will survive.

These bacteria are then purified and plated onto another culture medium.

Cloning the gene

The transformed bacteria are grown on nutrient rich media. This stimulates their
reproduction. As the bacteria reproduces, the gene will also be replicated. This
yields billions of copies of the gene of interest. Bacteriophage DNA makes roughly
102 copies of itself per day and E. coli replicates every thirty minutes (one E. coli
may have hundreds of plasmids).

INSULIN PRODUCTION

Insulin gene is isolated using the mRNA.

Plasmid and insulin gene are cut with the same restriction enzyme and combined
with DNA ligase.
Recombinant DNA is placed in the E. coli and the bacteria are stimulated to
reproduce and express the product of the gene, ie, insulin.
Insulin is removed, purified and packaged for human use.
 Human Growth Hormone, Insulin, Blood Thinners are developed via this
method.
 Resistance to certain conditions or diseases is conferred to plants by
employing recombinant DNA technology.
 KEY TERMS

 Recombinant DNA (rDNA) – dna formed from two different sources;

typically from different organisms. DNA ligase is used to join the two DNA
together into one genome.

 Transgenic Organisms- organisms altered by genetic engineering.

 Transformation- the introduction of foreign DNA to a bacterial cell.

 Vector – the transfer agent for foreign DNA. Carries the gene to the host.
May be bacterial plasmids or bacteriophages.
 Host – houses and replicates the rDNA. Hosts are able to grow quickly and
are easily manipulated.

USES OF GENETIC ENGINEERING

 Hormone production (insulin, HGH)

 Clean up of oil spills (bacteria are engineered to break down hydrocarbons
in oil.
 Disease resistance, increased yield, faster growth, predator resistance,
designer plants.
 Production of drugs from animals ( Herman, sheep)