DNA Technology Part 2

DNA Technology Isolation – of the DNA containing the required gene Insertion – of the DNA into a vector Transformation – Transfer of DNA into a suitable host Identification – finding those host organisms containing the vector and DNA (by use of gene markers) Growth/Cloning – of the successful host cells

Learning Objectives: Stage 3, 4 and 5 – Transformation, Identification and Cloning How is the DNA of the vector introduced into host cells? What are gene markers and how do they work?

Introduction of DNA into host cells – Transformation (stage 3) The plasmids must be reintroduced into the host cell e.g. bacteria This process is called transformation. 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.

Identification (stage 4) of bacteria containing the plasmid Only about 0.001% of bacterial cells take up any DNA/Plasmids when the two are mixed together. Firstly, we must identify the bacteria containing the plasmids – we do this by growing the bacteria on a medium containing an antibiotic. The antibiotic resistant gene is found in the plasmid only and therefore the bacteria that survive contain must contain the plasmid.

Identification (stage 4) of bacteria containing the plasmid with the DNA fragment Gene markers are used to identify which plasmids have taken up the DNA fragment. Gene markers can be: Resistance to an antibiotic A fluorescent protein An enzyme whose action can be identified Usually the gene marker is disrupted if the DNA fragment is present.

Fluorescent markers The gene from jellyfish which produces Green Fluorescent Protein (GFP) has been incorporated into a plasmid. If the DNA fragment has been inserted into the GFP gene, the bacterial will not glow and can be identified. If the DNA fragment has not been inserted into the GFP gene, the bacteria will glow and would not 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.

The Plasmid The ampicillin resistance gene is disrupted when the restriction enzymes cuts open the plasmid.

Antibiotic-resistance Markers The second antibiotic-resistance gene (e.g. resistance to ampicillin) 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 medium containing ampicillin. In order to identify these bacteria we use a process called replica plating.

Replica Plating The bacteria on the yellow plate have the plasmid. Ampicillin sensitive bacteria – these have the DNA fragment The bacteria on the yellow plate have the plasmid. The bacteria which do NOT grow on the green plate (containing ampicillin) contain a plasmid with a DNA fragment.

Cloning (stage 5) the bacteria Following successful identification of the bacteria containing the plasmid AND the DNA fragment, the bacteria are cloned. As the bacteria are cloned, so is the plasmid containing the DNA fragment. This type of gene cloning is in vivo (cloned within a living organism).