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Lecture #9Date _____ n Chapter 20~ DNA Technology & Genomics Slide 2 O.J. Simpson capital murder case,1/95-9/95 Odds of blood in Ford Bronco not being R. Goldmans: 6.5 billion to 1 Odds of blood on socks in bedroom not being N. Brown-Simpsons: 8.5 billion to 1 Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson: 21.5 billion to 1 Number of people on planet earth: 6.1 billion Odds of being struck by lightning in the U.S.: 2.8 million to 1 Odds of winning the Illinois Big Game lottery: 76 million to 1 Odds of getting killed driving to the gas station to buy a lottery ticket 4.5 million to 1 Odds of seeing 3 albino deer at the same time: 85 million to 1 Odds of having quintuplets: 85 million to 1 Odds of being struck by a meteorite: 10 trillion to 1 Slide 3 Recombinant DNA n Def: DNA in which genes from 2 different sources are linked n Genetic engineering: direct manipulation of genes for practical purposes n Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products Slide 4 Bacterial plasmids in gene cloning Slide 5 DNA Cloning n Restriction enzymes (endonucleases): in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific n Restriction site: recognition sequence for a particular restriction enzyme n Restriction fragments: segments of DNA cut by restriction enzymes in a reproducable way n Sticky end: short extensions of restriction fragments n DNA ligase: enzyme that can join the sticky ends of DNA fragments n Cloning vector: DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids) Slide 6 Restriction Enzymes Slide 7 Steps for eukaryotic gene cloning n Isolation of cloning vector (bacterial plasmid) & gene- source DNA (gene of interest) n Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase n Introduction of cloning vector into cells (transformation by bacterial cells) n Cloning of cells (and foreign genes) n Identification of cell clones carrying the gene of interest Slide 8 Cloning Slide 9 DNA Analysis & Genomics n PCR (polymerase chain reaction) n Gel electrophoresis n Restriction fragment analysis (RFLPs) n Southern blotting n DNA sequencing n Human genome project Slide 10 Polymerase chain reaction (PCR) n Amplification of any piece of DNA without cells (in vitro) n Materials: heat, DNA polymerase, nucleotides, single- stranded DNA primers n Applications: fossils, forensics, prenatal diagnosis, etc. Slide 11 DNA Analysis n Gel electrophoresis: separates nucleic acids or proteins on the basis of size or electrical charge creating DNA bands of the same length Slide 12 Restriction fragment analysis n Restriction fragment length polymorphisms (RFLPs) n Southern blotting: process that reveals sequences and the RFLPs in a DNA sequence n DNA Fingerprinting Slide 13 DNA Sequencing n Determination of nucleotide sequences (Sanger method, sequencing machine) n Genomics: the study of genomes based on DNA sequences n Human Genome Project Slide 14 Practical DNA Technology Uses n Diagnosis of disease n Human gene therapy n Pharmaceutical products (vaccines) n Forensics n Animal husbandry (transgenic organisms) n Genetic engineering in plants n Ethical concerns?