Recombinant DNA technology - A-Level Biology notes PDF

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Chapter 21 Recombinant DNA technology 21.1 Producing DNA fragments Recombinant DNA: DNA that has been formed artificially by combining constituents from different organisms. This results in the organism being transgenic / a genetically modified organism (GMO) The process of making a protein using the DNA technology of gene transfer and cloning involves a number of stages: 1. Isolation of the DNA fragments that have the gene for the desired protein 2. Insertion of the DNA fragment into a vector 3. Transformation of the host cells that have successfully taken up the gene by use of gene markers 4. Growth/cloning of the population of host cells. Before a gene can be transplanted it must be identified and isolated from the rest of the DNA. Ways to produce DNA fragments: - Conversion of mRNA to cDNA using reverse transcriptase - Using restriction endonucleases to cut fragments containing the desired gene from DNA - Creating the gene in a gene machine, usually based on a known protein structure Using reverse transcriptase to isolate a gene: Genetic information of a retroviruses is in form of RNA, but once they’re in a host cell they’re able to synthesis DNA from their RNA with the help of enzyme reverse transcriptase. 1. B-cells from islets of Langerhans in the human pancreas. As these cells are specialised to produce insulin, they make a lot of mRNA that codes 2. mRNA coding for insulin from B-cells for insulin 3. mRNA coding for insulin 4. mRNA acts as a template on which a single-stranded complimentary copy of DNA (cDNA) is formed using reverse transcriptase 5. Single-stranded cDNA is isolated by hydrolysis of the mRNA with an enzyme 6. Double-stranded DNA is formed on the template of the cDNA using DNA polymerase 7. Copy of human insulin gene

Using restriction endonuclease: Bacteria are often infected by viruses that inject their DNA into them to take over the cell. When this happens, some bacteria produce enzymes that cut up the viral DNA – restriction endonuclease.

Chapter 21 Recombinant DNA technology...