Transcript 5 In vivo gene cloning

DNA Technology
In vivo gene cloning
Learning Objectives:
1. Review knowledge of gene cloning so far.
2. Expand upon GCSE knowledge of genetic
engineering to understand the concept of
in vivo gene cloning.
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
Isolation of Required Gene
• Reverse transcriptase – what does it do?
• Restriction endonucleases – what do they
do?
Discuss with each other – 2 minutes!
Key terms: cDNA, sticky ends, blunt ends,
palindromic recognition sequence.
Isolation of Required Gene
Reverse transcriptase – makes a cDNA
copy of mRNA.
Restriction endonucleases – cut DNA at a
palindromic recognition sequence. May
produce DNA fragments with blunt ends
(non-overhanging bases) or sticky ends
(overhanging bases).
The PCR (Polymerase Chain
Reaction)
An in vitro method of gene cloning.
What ‘ingredients’ are needed to copy the
target DNA?
Primers, nucleotides, DNA polymerase.
The PCR (Polymerase Chain
Reaction)
What are the three stages of the PCR?
1. Heat to 95oC to break H-bonds
2. Cool to 60oC to allow primers to anneal
3. Heat to 72oC so DNA polymerase joins
the sugar-phosphate backbone between
new nucleotides as they H-bond to
complementary bases.
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 2 – Insertion in to a vector
• What is the importance of “sticky ends”?
• How can a DNA fragment be inserted into
a vector?
Importance of “sticky ends”
• DNA from different source can be joined
together IF they have the same sticky ends – the
same recognition site.
• In order to have the same sticky ends they must
have been cut using the same restriction
endonuclease.
• Sticky ends are joined together using DNA
ligase to join the sugar-phosphate
backbone together.
• The new DNA molecule is called
recombinant DNA.
Insertion of DNA into a vector
• VECTOR – any molecule/substance used
to transport DNA into a host cell.
• PLASMID – the most
commonly used vector.
A circular piece of DNA
found in bacteria.
Why?
• In vivo gene cloning.
• The plasmids will be reinserted into bacteria. As
the bacteria reproduce, they will copy the gene as
if it was a normal part of their DNA.
Example: The R Plasmid
• One of the antibiotic
resistant genes is
disrupted when the
restriction enzymes
cuts open the
plasmid.
• The other antibiotic
resistant gene is used
in selection of the
correct host cells.
Insertion into plasmids
What combinations of plasmid and donor
DNA could form?
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)
• Plasmids must be reintroduced into the
host cell (bacteria).
• This process is called transformation.
• Bacteria and plasmids are heated to 42oC
in a solution of calcium chloride.
• Even this well-used method is far from
perfect.
Few bacteria are transformed…
• Most cells don’t take up any plasmids.
• Some plasmids closed before the donor
DNA was inserted.
• Some of the DNA self-ligated.
Identification (stage 4) of
bacteria containing the plasmid
• Firstly, we must identify the bacteria containing
the plasmids – we do this by growing the
bacteria on a medium containing an antibiotic.
• We know to which antibiotics the bacteria should
be resistant. No plasmid = no resistance!
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
• GFP – green
fluorescent protein –
gene from jellyfish.
• Spliced into
plasmid.
• The restriction site
chosen is in the
middle of the GFP
gene.
Fluorescent markers
• If the donor DNA is added
correctly, the GFP gene is
disrupted and cells don’t
glow.
• If the donor DNA doesn’t
combine with the plasmid,
the GFP gene will be
intact and the cells will
glow.
Enzyme Markers
• Some plasmids contain
a gene for lactase.
• Lactase turns a
colourless substance
(X-gal) a blue colour.
• If the gene has been
disrupted by the
incorporation of the
gene fragment the
substrate will remain
colourless.
Example: The R Plasmid
• One of the antibiotic
resistance genes is
disrupted when the
restriction enzymes
cuts open the
plasmid.
• The other antibiotic
resistance gene is
used in selection of
the correct host cells.
Antibiotic-resistance Markers
• E.g. donor DNA may be
inserted at the Pst1 site.
• Bacteria with the correct
plasmid will be resistant
to tetracycline, but not
ampicillin.
• In order to identify these
bacteria we use a
process called replica
plating.
Replica Plating
Yellow
plate
contains
tetracycline
Ampicillin sensitive
bacteria – these have
the DNA fragment
Green
plate
contains
tetracycline
and
ampicillin
• Colonies are carefully transferred to the same
places on a new plate.
• 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).
Tasks
1. Without looking in the textbook, make a
list of the similarities and differences
between in vivo and in vitro gene cloning.
(You can check your answers afterwards
using p247!)
2. Answer the questions in the yellow box
on p 245-6.