Chapter 11: Gene Technology

1. Chapter 11: Gene Technology Biology II

2. History of Genetic Engineering • In 1973, Stanley Cohen and Herbert Boyer isolated genes that code for rRNA from DNA of African clawed frog • Inserted this DNA into the DNA of E.coli • E.coli produced frog rRNA during transcription, producing the first genetically altered organism.

3. Basic Steps of Genetic Engineering • Genetic engineering – the process of manipulating genes for practical purposes • May involve building recombinant DNA – DNA made from 2 or more different organisms • Can follow the steps of genetic engineering by examining how human gene for insulin is transferred into bacteria

4. Step 1: Cutting DNA • DNA of interest is cut by restriction enzymes • Bacterial enzymes that recognize and bind to specific short sequences of DNA and then cut the DNA between specific nucleotides within the sequences • Also cut is the vector – agent used to carry the gene of interest into another cell • Commonly used vectors include viruses, yeast, and plasmids – circular DNA molecules that can replicate independently of the main chromosomes of bacteria

5. Step 2: Making Recombinant DNA • DNA fragments from organism containing gene of interest are combined with DNA fragments from the vector • Enzyme called DNA ligase added to help bond ends of DNA fragments together

6. Step 3: Cloning • Many copies of the gene of interest are made each time the host cell reproduces in process called gene cloning • Bacteria reproduce by binary fission, producing identical offspring

7. Step 4: Screening • Cells that have received gene of interest are distinguished from those that did not take up the vector with the gene of interest

8. Restriction Enzymes: Cutting DNA • Restriction enzymes recognize a specific sequence of DNA • This sequence and the sequence on the complementary DNA strand are palindromes – they read the same forwards and backwards • Cuts of most restriction enzymes produce pieces of DNA with short single strands on each end, called sticky ends, that are complementary to each other

9. Making Recombinant DNA • Vectors used in genetic engineering contain only one nucleotide sequence that restriction enzyme recognizes • Vectors “open up” with same sticky ends as those of cut human DNA • 2 DNA molecules bond together through complementary base pairing of sticky ends

10. Screening of Engineered Cells • Cells that have taken up plasmid must be identified • Accomplished by growing bacteria on plates that contain the antibiotic tetracycline • Cells that have taken up the vectors contain the gene for tetracycline resistance and therefore survive on this medium • Surviving cells make copy of vector, eventually forming a colony of genetically identical cells, or clones

11. Confirmation of Cloned Genes • Surviving bacterial colonies must be tested for presence of gene of interest • The Southern Blot • DNA from each bacterial clone colony is isolated and cut into fragments by restriction enzymes • DNA fragments separated by gel electrophoresis • Uses electric field within gel to separate molecules by size

12. Gel Electrophoresis • Gel is rectangular slab of gelatin with line of rectangular wells near top edge • DNA sample placed into wells • DNA is negatively charged so it migrates towards positive pole when electric field is applied • Speed at which DNA fragments migrate down well determined by size (weight), with smallest moving the fastest

13. The Southern Blot (continued) • DNA bands transferred (blotted) directly onto filter paper • Filter paper moistened with probe solution – radioactive or fluorescent-labeled RNA or single-stranded DNA pieces that are complementary to the gene of interest • Only DNA fragments complementary to probe will bind and form visible bands

14. What’s Next? • Bacterial colonies containing gene of interest can be used in a variety of ways: • Isolate gene of interest to get pure DNA • Study evolution of gene by comparison across different organisms • Transfer isolated gene of interest to other organisms • Produce large quantities of protein coded for by gene for further study or to make drugs

15. Section 11-2 Human Applications of Genetic Engineering

16. The Human Genome Project • A research effort to sequence and locate the entire collection of genes in human cells • Many surprising findings: • Only 1-1.5% of DNA in human genome codes for protein • Human cells contain only 20,000-25,000 genes even though over 120,000 different forms of mRNA molecules had been counted

17. Genetically Engineered Drugs • Drugs can be manufactured by genetic engineering illnesses that result when body fails to make critical proteins • Factor VIII – protein that promotes blood clotting • Deficiency in factor VIII causes a type of hemophilia • Prior to GM medicines, hemophiliacs received blood factors isolated from donor blood, creating many potential risks for patients

18. Vaccines • Vaccine – solution containing all or part of a harmless version of a pathogen • Once injected, immune system recognizes pathogen’s surface proteins and responds by making antibodies • If future exposure occurs, antibodies are present to combat pathogen

19. Genetically Engineered Vaccines • Vaccines traditionally prepared using killed or weakened forms of pathogen • Potential to transmit disease if there is failure in process • GM vaccines are both effective and safe • Genes that code for surface protein of pathogen are inserted into DNA of harmless virus • Surface of modified virus display the surface proteins of the pathogen in addition to the virus’s own surface protein

20. RFLPs • No 2 individuals (except twins) have the same DNA sequence • When cut with restriction enzyme, length of resulting fragments will be different for each individual, called restriction fragment length polymorphism (RFLPs) • RFLPs used to identify individuals and determine relatedness among individuals

21. DNA Fingerprinting • DNA fingerprint – pattern of dark bands on photographic film made when an individual’s DNA restriction fragments are separated by gel electrophoresis, probed, and then exposed on X-ray film • Each individual (except twins) will have unique pattern of banding, or DNA fingerprint, since they have different RFLPs

22. Purposes of DNA Fingerprinting • To establish relatedness – paternity cases • Forensics – scientific investigation of of causes of injury and death • Identifying genes that cause genetic disorders

23. PCR • Polymerase Chain Reaction (PCR) – technique that makes many copies of selected segments of DNA • Useful when only a very small amount of DNA is available • Can produce a billionfold increase in DNA material within a few hours • Important for diagnosing genetic disorders and solving crimes, as well as for studying ancient fragments of DNA found in fossils

24. PCR Technique • Double-stranded DNA to be copied is heated, separating the strands • Short pieces of artificially made DNA called primers are added, binding to places of DNA where copying can begin • DNA polymerase and free nucleotides added, extending DNA by attaching complementary free nucleotides to primer • Process repeated, with the sample of DNA doubling every 5 minutes

25. Section 11-3 Genetic Engineering in Agriculture

26. Genetically Engineered Crops • Genetic engineers can change plants • Make plant more tolerable to drought conditions • Create plants that can adapt to different soils, climates, and environmental stresses • Create crops resistant to weedkiller glyphosate

27. Genetically Engineered Crops (con’d) • Develop crops resistant to insects • Insert gene isolated from soil bacteria that makes protein that injures gut of chewing insects • Make more nutritious crops • Add iron and beta carotene to rice

28. Risks of GM Plants • There are some potential problems with GM crops: • Weeds that have developed resistance to weedkillers • Allergies to products of introduced genes • Passing on introduced genes to wild relative • Ex. Wild corn • Pest resistant to GM toxins

29. Genetic Engineering in Farm Animals • Farmers use gene technology to improve or modify their farm animals: • Add Growth Hormone (GH) to supplement diet • Produced by bacteria into which the gene was introduced • Alter gene responsible for GH production in animals

30. Medically Useful Proteins • Genetic engineers have been able to add human genes to the genes of farm animals • Transgenic animal – animal with foreign DNA in their cells • Farm animals then produce human proteins in milk • Scientists can also clone animals in order to create herds that can make medically useful proteins

31. Cloning From Adult Animals • Ian Wilmut proved that animals could be cloned from differentiated cells of an adult animal • Lamb cloned from nucleus of mammary cell taken from adult sheep • Electric shock used to fuse mammary cells from one sheep with eggs cells lacking nuclei from another sheep • Fused cells divide to form embryos to be implanted into surrogate mothers

32. Problems With Cloning • Only a few cloned animals have survived for long • Some fatally oversized • Others have developmental problems • Genomic imprinting – chemical changes made to DNA prevent a gene’s expression without altering its sequence • Technical problems with this process, since normal process takes months for sperm and years for eggs