Transcript DNA_Technology_part2

DNA Technology
Part 2
DNA Technology
1. Isolation – of the DNA containing the
required gene
2. Insertion – of the DNA into a vector
3. Transformation – Transfer of DNA into a
suitable host
4. Identification – finding those host
organisms containing the vector and
DNA (by use of gene markers)
5. 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
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).