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DNA Applications

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by

Maria Averilla

on 6 March 2014

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Transcript of DNA Applications

Stage 1 - Isolation
This is the isolation of the DNA containing the required gene (e.g. insulin)

Reverse transcriptase and restriction endonuclease are used to create a complimentary DNA and cut the DNA into fragments.

Stage 2 - Insertion
This is when the recombinant DNA is inserted and into a vector.

The vector is used to transport DNA into a host cell.
Stage 3 - Transformation
This is the transfer of the DNA into a suitable host.
Stage 4 - identification
Using gene markers, we look for the host organism containing the vector and DNA
Stage 5 - Growth/ Cloning
After marking the successful host cells, they are cultured to produce the protein on a large scale.
DNA Applications
DNA Technology

Using reverse transcriptase
B-cells from islets of Lagerhans in the human pancreas
Extract mature mRNA coding for Insulin
A single stranded complimentary copy of DNA (cDNA) is formed using reverse transcriptase on the mRNA template
Single stranded cDNA is used to form a double stranded DNA usingn DNA Polymerase
This forms a double stranded copy of the human insulin gene
They are used to catalyse the production of viral RNA to DNA so that it can be transcribed by the host cell into proteins.

The DNA is known as complimentary DNA (cDNA) because it is made up of the nucleotides that are complimentary to the mRNA.
Insertion of DNA fragment of a vector
Importance of 'sticky ends'
DNA from different source can be joined together if they have the same recognition site, cut by the same restriction endonuclease.

Sticky ends are joined together using DNA Ligase to join the phosphate-sugar framework.

The new DNA molecule is called recombinant DNA
Plasmids
One of the antibiotic resistant genes is disrupted when the restriction enzyme cuts open the plasmid.

The other antibiotic resistant gene is used in the selection of the correct host cell
Vector - Used to transport DNA into a host cell.

Plasmid - a circular piece of DNA found in a bacteria

Plasmids are always useful because they nearly always contain antibiotic resistance gene and restriction endonuclease is used to break the loop.
Plasmid must be reintroduced into host cell e.g. bacteria

The bacteria, plasmids and calcium are mixed together.

By altering the temperature, the bacteria become permeable and the plasmid can pass through the cell membrane
Bacteria containing the plasmid
Only about 0.001% of bacterial cells take up any DNA/ Plasmids when they are mixed together.

We must identify the bacteria by growing the bacteria on a medium containing an antibiotic

The antibiotic gene is found in the plasmid only and therefore the bacteria that survive must contain the plasmid.
Gene markers
Used to identify which plasmids have taken up the gene.

Can be:
resistance to an antibiotic
fluorecent protein
identifiable enzyme action

This is disrupted if the DNA fragment is present.
Fluorescent markers
The gene from jellyfish produces green fluorescent protein (GFP) has been incorporated into a plasmid.

If the DNA fragment has been inserted into the GFP gene, the bacteria will not glow and can be identified.

If the DNA fragment has not been inserted into the GFP gene, the bacteria will glow and wouldn't be used.
Enzyme markers
The enzyme lactase turns a colourless substance, a blue colour.

If the gene has been disrupted by the incorporation of the gene fragment, the substrate will remain colourless.
Antibiotic resistance markers
The second anitbiotic-resistance gene (e.g. ampicillin resistance) is used to identify those plasmids with a DNA fragment in them.

If the DNA fragment has been inserted into the ampicillin resistance gene, it will no longer grow on a medium containing ampicillin.

In order to identify these bacteria, we use a process called replica plating.
Replica plating
Ampicillin sensitive bacteria – these have the DNA fragment

The bacteria on the yellow plate have the plasmid.

The bacteria which don't grow on the green plate (containing ampicillin) contain a plasmid with a DNA fragment.
Restriction endonuclease
Bacteria containing restriction enzymes in order to protect themselves from invading viruses.

Restriction enzymes are used by bacteria to cut up the viral DNA in a staggered fashion.

These enzymes cut DNA at specific bases called a recognition sequence.
Blunt ends
This is when the cut occurs between two opposite base pairs, leaving two straight edges.
Most restriction enzymes make a staggered cut in two chains, forming sticky ends.
Sticky ends
Have a strand of single stranded DNA, which are complimentary and palindronic.
Cut in a staggered fashion
It will join another sticky end if it has been cut with the same restriction endonuclease.
Following the successful identification of the bacteria containing the plasmid and the DNA fragment, the bacteria is collected.

As the bacteria are cloned, so is the plasmid containing the DNA fragment

This type of gene is in vivo (cloned within a living organism)

Maria Averilla
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