Gene Technology1 Biotechnology You are only responsible for the material we get through in class End of Chapter questions: Understand: 1,2,4,5,7, Apply: all Synthesize: 1 Do all mQuiz questions Chapter 17

Gene Technology2 What are the methods of genetic engineering ? DNA cleavage Restriction endonucleases “sticky ends”

Gene Technology3 — + Gel electrophoresis Used to analyze DNA fragments Agarose – acts like a sieve DNA has negative charge

Gene Technology4 How can one determine which bands have a particular sequence? Southern blotting -- involves 3 key steps 1. Transfer to nitrocellulose 2. “Probe” with 32 P labeled DNA sequence (e.g., “AATGGCAGGT”) 3. Perform autoradiography

Gene Technology5 DNA fragments are manipulated (e.g., ‘cloned’) by inserting into a ‘vector’ A ‘vector’ is a carrier -- Plasmids or viruses “Cloning” means to copy Plasmids with special genes are used

Gene Technology6 How can a “genomic library” be created? -- Cells with DNA fragments from entire genome 1. Cut up DNA into pieces 2. insert pieces at random into vector 3. “Transfect” into bacteria Problems, however: -- Few bacteria receive a plasmid -- Not all plasmids contain a DNA fragment Also… Very few plasmids contain DNA with any particular gene Cloning Steps

Gene Technology7 How to screen for recombinant plasmids? Cells grown on Medium containing ampicillin and Xgal Form blue or white colonies

Gene Technology8 How to screen for cells containing a specific DNA fragment? Plate library Make replica on Nitrocellulose Probe with Radio-labeled “DNA probe” Perform autoradiography Find original colony

Gene Technology9 How can the DNA for a particular gene be isolated from original organism? -- one way is a “cDNA” (complementary DNA) Starts with mRNA Reverse transcriptase DNA polymerase cDNA lacks intron sequences

Gene Technology10 What can cloned genes be used for? Transferring to other organisms, e.g. -- insulin production to bacteria -- herbicide/ insect resistance to plants -- crop yield & nutritional value (e.g. ‘golden rice’) -- biofuel (e.g., rape seed oil) Gene therapy … some day -- cystic fibrosis -- hemophilia -- various cancers -- many others Gene sequencing -- evolutionary relationships -- protein structure predications Sheep engineered to produce the human protein alpha-1-antitrypsin (AAT) in milk for treatment of emphysema Control vs Roundup resistant alfalfa in field treated with Roundup

Gene Technology11 “DNA Fingerprinting” Used to identify source of DNA Small genetic differences create Restriction Fragment Length Polymorphisms (RFLPs) Forensic samples (‘CSI Biology’) -- blood, semen, saliva How are RFLPs used to create a DNA fingerprint? DNA fingerprint

Gene Technology12 35 cycles = 2 35 = 68 billion copies PCR PCR is used to analyze very small amounts of DNA PCR = Polymerase Chain Reaction Requires: Thermocycler Primers Taq-polymerase PCR animation