DNA and Gene
Cloning
- DNA revision
- Gene cloning
- cDNA and reverse transcriptase
- Restriction endonucleases
Collect some nucleotides
• The 5’ prime and 3’ prime ends of the bases
must be the right way round!
Collect the following:
•
•
•
•
•
•
•
•
3 yellow bases with a phosphate and a black sugar
3 yellow bases with a phosphate and a red sugar
3 green bases with a phosphate and a black sugar
3 green bases with a phosphate and a red sugar
4 blue bases with a phosphate and a black sugar
4 blue bases with a phosphate and a red sugar
4 orange bases with a phosphate and a black sugar
4 orange bases with a phosphate and a red sugar
Build:
• With nucleotides containing black sugars:
Blue
Blue
Orange
Yellow
Yellow
Green
Orange
Join some nucleotides together to
form some DNA.
• Green (Guanine) pairs
with yellow (Cytosine)
• Blue (Adenine) pairs
with orange (Thymine)
In your group and using the model…
Describe how DNA
replicates
Key terms:
- DNA helicase
- DNA polymerase
- Complementary bases
Describe how DNA is
used as a template for
protein production
Key terms:
- DNA helicase
- RNA polymerase
- Complementary bases
- Transcription
- Translation
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
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 1 – Producing DNA fragments
• How is complementary DNA made using
reverse transcriptase?
• How are restriction endonucleases used
to cut DNA into fragments?
Reverse Transcriptase
• A group of viruses called retroviruses (e.g.
HIV) contain an enzyme called reverse
transcriptase.
• It is used to turn viral RNA into DNA so
that it can be transcribed by the host cell
into proteins.
Producing DNA copies from mRNA
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Reverse Transcriptase
DNA
polymerase
• Reverse transcriptase makes DNA from
an RNA template – it does the opposite of
transcription.
Using reverse transcriptase
β-cells from Islets of Langerhans in the human pancreas.
Extract mature mRNA coding for insulin.
A single stranded complementary DNA strand (cDNA) is
formed using reverse transcriptase and the mRNA
template.
Single stranded cDNA is used to form double stranded
DNA using DNA polymerase.
This forms a double stranded copy of the human insulin
gene.
Restriction Enzymes
• Bacteria contain restriction enzymes in
order to protect themselves from invading
viruses.
• Restriction enzymes are used by bacteria
to cut up the viral DNA.
• These enzymes cut DNA at specific sites –
this property can be useful in gene
technology.
Restriction Endonucleases
• Have highly specific active sites.
• Cut DNA at specific sites – about 4-8 base pairs
long – these are called recognition sites.
• Recognition sites are palindromic - the
sequence and its complement are the same but
reversed.
• E.g.
-GAATTC-CTTAAG-
Assemble the following sequence:
With any colour of sugar:
5’ – GTCGACCCGGGTCGACA – 3’
Then complete the complementary strand.
Don’t forget to orient it correctly!
G = green
C = yellow
A = blue
T = orange
Restriction enzymes
• Sma1 cuts between the C and the G of
CCCGGG.
• Taq1 cuts between the T and the C of TCGA.
• What do you notice when you perform each
of these cuts?
• What if you use both enzymes?
Producing DNA copies by cutting DNA
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Restriction Enzymes – “Blunt
Ends”
Some restriction
enzymes cut
straight across
both chains
forming blunt
ends.
Restriction Enzymes – “Sticky
Ends”
• Most restriction enzymes make a staggered cut
in the two chains, forming sticky ends.
Sticky Ends…
• Sticky ends have a strand of single
stranded DNA which are complementary
to each other.
• They will join with another sticky end but
only if it has been cut with the same
restriction enzyme.
How do we know which section of
DNA to use?
• We use a process called Southern blotting.
• Involves gel electrophoresis, binding of DNA to
a membrane, and visualisation of the DNA
section of interest using a DNA probe.
Animation
Gel Electrophoresis
• A sample containing DNA fragments of
different sizes is placed at one end of an
agarose gel.
• A current is passed through the gel, causing
negatively-charged DNA (due to the
phosphate groups) to be attracted to the
positive electrode.
• Smaller fragments move faster and further
than larger fragments.
Southern Blotting
• The gel is treated with a basic solution to
separate the DNA strands (breaks the Hbonds).
• A nylon membrane is placed over the gel and
many paper towels are placed on top.
• Over many hours, the solution is drawn up
into the paper towels, bringing the DNA with
it. The DNA sticks to the membrane.
DNA Hybridisation
• A radioactively-labelled DNA probe (single
stranded DNA with a sequence
complementary to the section of interest) is
washed over the membrane.
• The probe binds with any fragments of DNA
containing the complementary base
sequence.
• X-ray film is used to detect the presence of
hybridised DNA fragments.
Restriction mapping
• After gel electrophoresis has been performed,
we may wish to work out in what order
different fragments appear in the DNA
sample.
• A restriction map shows us where the
fragments are located.
Locating genes
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Locating a specific gene
One approach to locating genes is to use a DNA probe.
This is a short section of DNA that has been labelled, for
example with radioactive phosphorous or a fluorescent marker.
The DNA is of a known sequence corresponding to the
gene being looked for, for example the cystic fibrosis
gene during clinical screening.
fluorescent
marker
DNA probe
target gene
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Using a gene probe
Before using a gene probe the DNA needs to be heated to
separate the two strands.
The temperature is then
reduced so that the probe
can ‘anneal’ or hybridize
with the sample DNA as a
result of complementary
base pairing.
hybridized probe
The location of the gene is then identified. The method
used depends on the method of labelling. Radioactive tags
are located by exposing the DNA to a photographic plate;
fluorescent tags are located by using UV light in a
fluorescent microscope.
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Carry out the ‘Restriction mapping and
Southern blotting’ activity.