Genetic Engineering

Página 1

Genetic Engineering

"Just because we can, does it mean we should?"

The principles of genetic engineering

Useful genes isolated from one organism and instead into another organism creating recombinant DNA.

4 Stages: 1. Obtain gene, 2. Gene place in vector, 3. Vector carries gene into cell, 4. Cell expresses the novel gene.

1. Obtaining the required gene

mRNA obtained from cells where the gene is expressed.

Reverse transcription forms a single strand of cDNA from mRNA. Go from RNA to complementary DNA.

If the nucleotide sequence is known, we can use automated polynucleotide synthesiser. (Alternative way)

If gene sequence is known, PCR is used. (3rd Method).

A DNA probe can be located gene to cut out.

2. Placing the gene into a vector

Plasmids are mixed with the restriction enzymes.

Plasmids cut at specific sites by enzymes.

Plasmid has exposed bases - "sticky ends".

Free nucleotides complementary to sticky ends are added to the gene to be added.

DNA ligase then anneals the gene.

Or gene sealed in a weakened virus.

To carry DNA into cell (Bacteriophage).

3. Getting vector into recipient cell

There are many methods to do this:

Heat shock treatment - periods of cold and heat and calcium chloride. This creates holes in the membrane.

Electroporation - High voltage pulse.

Electrofusion - Electric fields.

Transfusion - DNA put into bacteriophage.

T1 plasmid - into Agrobacterium tumefaciens naturally infect some plants inserting genome.

Gene gun - Attaches genes onto platinum and fires it into a bacterium.

Restriction enzymes

E.g. ECOR1 - Isolated from E.coli (Bacterium).

They come from bacteria as some bacteria produce enzymes.

They recognise specific sequences of DNA and cut the molecule there.

ECOR1 cuts between G and A.

Leave either blunt or sticky ends.

All restriction enzymes come from bacteria.

Ligase enzymes

Join DNA fragments

Join sugar phosphate backbone.

An example of a major scientific success is the human insulin production.