1. Option F: Microbes and Biotechnology F.3 Microbes and Biotechnology

2. F.3.1 State that reverse transcriptase catalyses the production of DNA from RNA

3. F.3.2 Explain how reverse transcriptase is used in molecular biology Reverse transcriptase (RT) is obtained from retroviruses Used to make DNA/genes for gene transfer, gene therapy, or gene libraries DNA can be made from mature mRNA – this means making DNA without introns cDNA (Complementary DNA) is made using RT and dsDNA is made using DNA polymerase Example: human insulin What can it be used for? Cloning DNA Diagnosing microbial diseases

4. Task: Put these sentences in order DNA polymerase is used to synthesise the complimentary strand to make dsDNA Reverse transcriptase produces cDNA The bacteria reproduce and make insulin Mature mRNA is made that is complimentary to the insulin gene Insulin is harvested This dsDNA is isolated DNA is taken from a pancreatic cell (β cells) Bacterial cells are stimulated to take up genetically engineered plasmid Isolated dsDNA is inserted into a bacterial plasmid

5. Task: Put these sentences in order DNA is taken from a pancreatic cell (β cells) Mature mRNA is made that is complimentary to the insulin gene Reverse transcriptase produces cDNA DNA polymerase is used to synthesise the complimentary strand to make dsDNA This dsDNA is isolated Isolated dsDNA is inserted into a bacterial plasmid Bacterial cells are stimulated to take up genetically engineered plasmid The bacteria reproduce and make insulin Insulin is harvested

6. F.3.3 Distinguish between somatic and germ-line therapy Gene therapy: introduction of new genes into human cells to alleviate disease. These genes are delivered by vectors (i.e. viruses). Somatic therapy: affects only body cells (Single-gene defects: Cystic fibrosis or haemophilia) Germ-line therapy: change germ cell (gamete) DNA What are the major differences between the two then?

7. F.3.4 Outline the use of viral vectors in gene therapy Gene therapy involves replacement of defective genes Virus vectors are genetically modified to be safely used The desired gene/allele is inserted into the viral genome Target somatic cells are removed and infected with virus. Desired gene is inserted into target cells Cells are replaced in patient so desired genes can be expressed. Example: Severe combined immune deficiency disease (SCID) with defective enzyme called ADA

8. F.3.5 Discuss the risks of gene therapy May trigger immune response to virus in patient Unknown long-term effects Impact on offspring of treated person is unknown Risk of ‘designer babies’ Risk of consent during research trials being uninformed Virus may become infectious Gene may not be inserted into correct region of DNA (mutation) May infect other cells besides target cells