Chapter 14 Biotechnology and Genomics DNA Cloning Biotechnology
Chapter 14 Biotechnology and Genomics
DNA Cloning: Biotechnology and Genomics 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. 2
Biotechnology and Genomics DNA Cloning: Recombinant DNA Technology Recombinant DNA (r. DNA) contains DNA from two or more different sources, such as a human cell and a bacterial cell This requires: 1. A vector w This is a means to transfer foreign genetic material into a cell. w Plasmids (small accessory rings of DNA from bacteria) are common vectors 3
DNA Cloning: Plasmids Biotechnology and Genomics 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 4
Biotechnology and Genomics DNA Cloning: Recombinant DNA Technology Recombinant DNA technology A set of techniques for combining genes from different sources and then transferring the resulting r. DNA into cells. 1. Plasmids have been very useful in this endeavor 2. Viruses have also been used for this purpose 5
Biotechnology and Genomics DNA Cloning: Recombinant DNA Technology 6 How to create recombinant DNA (r. DNA)? 1. You must insert one organism’s DNA into the vector DNA 2. To do this you need the following: w A restriction enzyme – which cleaves, or cuts, DNA w A DNA ligase enzyme seals DNA into the DNA of the vector
DNA Cloning: Restriction Enzymes Biotechnology and Genomics Restriction Enzymes 1. These are “cutting” tools for making r. DNA 2. They are actually bacterial enzymes called endonucleases. w They are produced by bacteria to protect themselves from invading viral DNA w They work by finding and chopping out the foreign DNA. 7
Biotechnology and Genomics Restriction Enzyme Videos Restriction Endonucleases http: //highered. mcgraw-hill. com/olc/dl/120078/bio 37. swf 8
DNA Cloning: Restriction Enzymes Biotechnology and Genomics 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”. w These single stranded ends of DNA can base pair with any other piece of DNA which was cut with the same restriction enzyme. 9
DNA Cloning: Restriction Enzymes Biotechnology and Genomics 10
Cloning a Human Gene 11
DNA Cloning: Creating Custom Bacteria Biotechnology and Genomics 12 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
Cloning a Human Gene 13
DNA Cloning: Creating Custom Bacteria Biotechnology and Genomics 14 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
Cloning a Human Gene 15
DNA Cloning: Creating Custom Bacteria Biotechnology and Genomics 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 16
Cloning a Human Gene 17
DNA Cloning: Creating Custom Bacteria Biotechnology and Genomics 18 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
DNA Cloning: Steps in Cloning a Gene Biotechnology and Genomics 19 Steps in Cloning a Gene http: //highered. mcgraw-hill. com/olc/dl/120078/micro 10. swf
Cloning a Human Gene 20
Early Genetic Engineering Experiment Biotechnology and Genomics Early Genetic Engineering Experiment http: //highered. mcgraw-hill. com/olc/dl/120078/bio 38. swf 21
Biotechnology and Genomics 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 m. RNA from these cells that codes for insulin 3. Use the enzyme, reverse transcriptase, to make DNA from the m. RNA. ◊ This is called complementary DNA, c. DNA
Biotechnology and Genomics DNA Cloning: c. DNA Video http: //highered. mcgraw-hill. com/olc/dl/120078/bio_h. swf 23
Biotechnology and Genomics DNA Cloning: Polymerase Chain Reaction (PCR) 24 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
Biotechnology and Genomics DNA Cloning: Polymerase Chain Reaction (PCR) Steps of PCR technique: 25 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 (> 94 o. C) is applied to test tube 3. H bonds break & DNA splits 4. Cool down test tube; DNA replicates 5. Repeat steps over and over
Biotechnology and Genomics Video Polymerase Chain Reaction (PCR) 26 Polymerase Chain Reaction Video http: //highered. mcgraw-hill. com/olc/dl/120078/micro 15. swf
PCR 27
Applications of PCR: Analyzing DNA Segments Biotechnology and Genomics 28 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
Gel Electrophoresis Biotechnology and Genomics 29 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
Gel Electrophoresis Biotechnology and Genomics 30 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
Restriction Length Fragment Polymorphisms Video Biotechnology and Genomics Restriction Length Fragment Polymorphisms Video http: //highered. mcgraw-hill. com/olc/dl/120078/bio 20. swf 31
DNA Fingerprinting & Paternity 32
Gel Electrophoresis Biotechnology and Genomics 33 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.
Biotechnology Products: Transgenic Bacteria Biotechnology and Genomics 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) 34
Genetically Engineered Bacteria 35
Biotechnology and Genomics Biotechnology Products: Transgenic Plants and Animals 36 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 (b. GH) The procedure has been used to produce larger fishes, cows, pigs, rabbits, and sheep
Biotechnology and Genomics 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: w Cystic fibrosis w Cancer w Blood diseases, etc. 37
Transgenic Mammals 38
Biotechnology and Genomics 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.
Biotechnology and Genomics 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
Gene Therapy 41
Biotechnology and Genomics Gene Therapy • In Vivo Gene Therapy ◊ Genes inserted in human body, via nasal sprays, viruses, or liposomes Examples: Cystic Fibrosis treatments Cancer therapies 42
Biotechnology and Genomics See Human Genome Project Power. Point Presentation 43
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