dna technology. 1.isolation – of the dna containing the required gene 2.insertion – of the dna...
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DNA Technology
1. Isolation – of the DNA containing the required gene
2. Insertion – of the DNA into a vector3. Transformation – Transfer of DNA into a
suitable host4. 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
• Reverse Transcriptase makes DNA from an RNA template – it does the opposite of transcription.
DNA polymerase
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) isformed using reverse transcriptase on the mRNA template.
Single stranded cDNA is used to form double strandedDNA using DNA polymerase
This forms a double stranded copy of the Human Insulingene.
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)
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