Download Using reverse transcriptase

DNA Technology
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.
Reverse Transcriptase
DNA
polymerase
• Reverse Transcriptase makes DNA from
an RNA template – it does the opposite of
transcription.
Using reverse transcriptase
B-cells from Islets of Langerhans in the Human pancreas.
Extract mature mRNA coding for Insulin.
A single stranded complementary copy of DNA (cDNA) is
formed using reverse transcriptase on 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 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.
Restriction Enzymes
• Also called restriction endonucleases.
• Have highly specific active sites.
• Usually cut DNA at specific sites – about 4-8
base pairs long – these are called recognition
sites.
• Recognition sites are usually palindronic, which
means the sequence and its complement are
the same but reversed.
• E.g. GAATTC and the complement CTTAAG
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 – used to transport DNA into a
host cell.
• PLASMID – the most commonly used
vector. A circular piece of DNA found in
bacteria.
• Plasmids are useful because the nearly
always contain antibiotic resistance genes
(see later).
The 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.
(See later)
Insertion into plasmids
• What combinations of plasmid will form?