Unit 5.6 Recombinant DNA Technology
- Created by: theviahsiva
- Created on: 15-05-15 11:07
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- Recombinant DNA Technology
- Method
- 1) Isolation of gene and vector
- 2) Insertion of gene into vector
- 3)Transformation of vector into cells
- 4) Growth
- 5) Selection of cells with genes
- Reverse Transcriptase
- 1) Extract mRNA
- 2) Add reverse transcriptase and cDNA will be made
- 3) Add DNA polymerase to make double stranded DNA
- 4) Insert DNA into vector
- Insulin Production
- 1) Insulin gene is located using a gene probe and isolated
- 2) Same restriction enzymes used to cut DNA of
the plasmid and the insulin gene.
- Forms complementary sticky ends
- 3) sticky ends anneal and DNA ligase joins the DNA pieces
- 4)The plasmid is transformed into E.coli bacteria
- 5)E.coli grown in the presence of an antibiotic.
- marker gene (antibiotic resistance) in the plasmid.
- so cells that have taken in the plasmid are resistant to antibiotics
- marker gene (antibiotic resistance) in the plasmid.
- 6) E.coli cloned and insulin extracted
- Adv
- Complex proteins produced in large quantities
- Don’t need to extract proteins from mammalian organs
- Disadv
- expensive for large scale production
- Difficult to identify/isolate correct gene
- More than 1 gene may be needed for complex proteins
- Disruption of normal genes during insertion
- Transfer of antibiotic resistant genes into E.coli in human gut
- Method
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