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DNA Cloning:

DNA Cloning:. Gene Cloning is the production of genetically identical copies of DNA, cells, or organisms. Can be done to: ♦ Create many identical copies of the same gene ( gene cloning )

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DNA Cloning:

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  1. Biotechnology and Genomics

  2. DNA Cloning: • Gene Cloning is the production of genetically identical copies of DNA, cells, or organisms. • Can be done to: ♦ Create many identical copies of the same gene (gene cloning) ♦ Genetically modify organisms in a beneficial way ● When used to modify a human it is called gene therapy. ● When used to modify another organism the new organisms created are called transgenic organisms.

  3. DNA Cloning:Recombinant DNA Technology • Recombinant DNA (rDNA) contains DNA from two or more different sources, such as a human cell and a bacterial cell • This requires: 1. A vector • This is a means to transfer foreign genetic material into a cell. • Plasmids (small accessory rings of DNA from bacteria) are common vectors

  4. DNA Cloning:Plasmids • There are many kinds of plasmids • R plasmids carry genes for enzymes that destroy antibiotics: 1. Thus, bacteria that carry these plasmids and their genes are resistant to antibiotics ●This means they are not killed by the antibiotic. ● This is a serious problem for humans as more and more bacteria that cause human diseases are becoming resistant to our antibiotics

  5. DNA Cloning:Recombinant DNA Technology • Recombinant DNA technology • A set of techniques for combining genes from different sources and then transferring the resulting rDNA into cells. • Plasmids have been very useful in this endeavor • Viruses have also been used for this purpose

  6. DNA Cloning:Recombinant DNA Technology • How to create recombinant DNA (rDNA)? • 1. You must insert one organism’s DNA into the vector DNA • 2. To do this you need the following: • A restriction enzyme – which cleaves, or cuts, DNA • A DNA ligase enzyme - seals DNA into the DNA of the vector

  7. DNA Cloning:Restriction Enzymes • Restriction Enzymes • 1. These are “cutting” tools for making rDNA • 2. They are actually bacterial enzymes called endonucleases. • They are produced by bacteria to protect themselves from invading viral DNA • They work by finding and chopping out the foreign DNA.

  8. Restriction Enzyme Videos Restriction Endonucleases http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf

  9. DNA Cloning:Restriction Enzymes • How do restriction enzymes work? • 1. They recognize short nucleotide sequences in the DNA and cut at very specific points • 2. They produce staggered cuts which are called “sticky ends”. • These single-stranded ends of DNA can base-pair with any other piece of DNA which was cut with the same restriction enzyme.

  10. DNA Cloning:Restriction Enzymes

  11. Cloning a Human Gene

  12. DNA Cloning:Creating Custom Bacteria • Steps to make custom bacteria: • 1. Obtain 2 kinds of DNA: • a. Human DNA of interest • Example: gene for human insulin • b. Bacterial plasmid • This will act as a vector to get human DNA into a bacterium

  13. Cloning a Human Gene

  14. DNA Cloning:Creating Custom Bacteria • 2. Treat plasmid and human DNA with the same restriction enzyme • a. Pick a restriction enzyme that: • • Cuts the plasmid in only ONE location (just to open up the plasmid) • • Cuts the human DNA in just TWO spots • ◊ Just in front of the gene for insulin • ◊ Just behind of the gene for insulin

  15. Cloning a Human Gene

  16. DNA Cloning:Creating Custom Bacteria • 3. Mix the human DNA with the cut plasmid • a. The sticky ends of the plasmid should base-pair with the complementary sticky ends of the human DNA • b. Use DNA ligase to join the two DNA molecules together with covalent bonds • c. The result is recombinant DNA

  17. Cloning a Human Gene

  18. DNA Cloning:Creating Custom Bacteria • 4. Mix the recombinant DNA plasmid with bacteria • a. Given the right conditions, the bacteria take up the plasmid DNA by a process known as transformation • 5. The bacteria then reproduce asexually and “clone” themselves. • a. All of these cloned bacteria will have the gene for human insulin • ◊ They will now produce insulin for us

  19. DNA Cloning:Steps in Cloning a Gene Steps in Cloning a Gene http://highered.mcgraw-hill.com/olc/dl/120078/micro10.swf

  20. Cloning a Human Gene

  21. Early GeneticEngineering Experiment Early Genetic Engineering Experiment http://highered.mcgraw-hill.com/olc/dl/120078/bio38.swf

  22. DNA Cloning:How to get the gene for human insulin? • Gene given to the bacteria must NOT contain introns. How do you get intron free DNA? • 1. Obtain cells from tissues that produce a lot of the desired protein • a. In this case, get pancreatic cells • 2. Isolate the mRNA from these cells that codes for insulin • 3. Use the enzyme, reverse transcriptase, to make DNA from the mRNA. • ◊ This is called complementary DNA, cDNA

  23. DNA Cloning:cDNA Video cDNA Video http://highered.mcgraw-hill.com/olc/dl/120078/bio_h.swf

  24. DNA Cloning:Polymerase Chain Reaction (PCR) • Technique to create copies of pieces of DNA quickly in a test tube. It is said to amplify a targeted sequence of DNA • Allows thousands of copies to be made of small samples of DNA • Requires: • DNA polymerase • A supply of nucleotides for the new DNA strands

  25. DNA Cloning:Polymerase Chain Reaction (PCR) • Steps of PCR technique: • 1. DNA to be copied is mixed with heat- resistant versions of DNA polymerase and loose nucleotides • a. These were discovered in the bacterium, Thermus aquaticus, which lives in hot springs. • 2. High heat (> 94oC) is applied to test tube • 3. H-bonds break & DNA splits • 4. Cool down test tube; DNA replicates • 5. Repeat steps over and over

  26. VideoPolymerase Chain Reaction (PCR) Polymerase Chain Reaction Video http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

  27. PCR

  28. Applications of PCR:Analyzing DNA Segments • DNA can be subjected to DNA fingerprinting • Treat DNA segment with restriction enzymes • This creates a unique collection of different fragments which differ from each other based on their lengths. (RFLPs = restriction fragment length polymorphisms) • Gel electrophoresis separates the fragments according to their charge/size • Produces distinctive banding pattern

  29. Gel Electrophoresis • Gel electrophoresis is a method of physically sorting macromolecules (DNA or proteins) • Steps of procedure: 1. Samples of DNA are placed in wells at one end of flat, rectangular gel 2. Gel is placed in an box with + and - electrodes at each end a. DNA, (-) charged, is placed at (-) end b. Electric current pulls the DNA thru gel towards the (+) electrode

  30. Gel Electrophoresis • 3. As pieces of DNA move through the gel, they will separate according to their size: • a. Smaller pieces move more quickly and end up traveling further through the gel • b. Larger pieces will be bogged down in gel and not move as far in the same amount of time • 4. You end up with “bands” in each lane of the gel. • a. Each band represents a different size DNA

  31. Restriction Length Fragment Polymorphisms Video Restriction Length Fragment Polymorphisms Video http://highered.mcgraw-hill.com/olc/dl/120078/bio20.swf

  32. DNA Fingerprinting & Paternity

  33. Gel Electrophoresis • 5. If DNA from different people is run thru the same gel, differences and similarities can be studied • 6. DNA fingerprinting is used to: • a. Measure number of repeats of short sequences of DNA. People differ in the number of these repeats. • b. Used in paternity suits, rape cases, corpse ID, identification of viral infections, identifying people with genetic disorders, detection of cancer, identification of trade in endangered species .

  34. Biotechnology Products:Transgenic Bacteria • Transgenic organisms have had a foreign gene inserted into their genetic make-up • Transgenic bacteria now produce: • Insulin, Human Growth Hormone, clotting factor VIII, hepatitis B vaccine • Oil-Eating Bacteria • Promote plant health (Ice-minus strawberries)

  35. Genetically Engineered Bacteria

  36. Biotechnology Products:Transgenic Plants and Animals • Transgenic Plants have been created to produce: • Pest resistant agricultural crops • Human hormones • Transgenic Animals • Many types of animal eggs have taken up the gene for bovine growth hormone (bGH) • The procedure has been used to produce larger fishes, cows, pigs, rabbits, and sheep

  37. Biotechnology Products:Transgenic Plants and Animals • Gene Pharming: • Use of transgenic farm animals to produce pharmaceuticals • Genes coding for therapeutic & diagnostic proteins are incorporated into an animal’s DNA • The proteins appear in the animal’s milk • Plans are to produce drugs to treat: • Cystic fibrosis • Cancer • Blood diseases, etc.

  38. Transgenic Mammals

  39. Human Genome Project • Genome - All the genetic information of an individual (or species) • Goals of Human Genome Project: • Determine the base pair sequence of human DNA • Launched in 1990; completed a working draft in 2003 • Construct a map showing sequence of genes on specific chromosomes (approximately 25,000 genes code for proteins) • Other species sequences are being determined.

  40. Gene Therapy • The insertion of genetic material into human cells for the treatment of a disorder • Ex Vivo Gene Therapy ◊ Genes combined outside of body and then placed inside Examples: - Children with Severe Combined Immunodeficiency injected with modified bone marrow stem cells

  41. Gene Therapy

  42. Gene Therapy • • In Vivo Gene Therapy • ◊ Genes inserted in human body, via nasal sprays, viruses, or liposomes • Examples: - Cystic Fibrosis treatments - Cancer therapies

  43. See Human Genome Project PowerPoint Presentation

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