1 / 43

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 )

bwhitehead
Download Presentation

DNA Cloning:

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related