Lecture 9, Ch. 20

Lecture #9

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Chapter 20~ DNA Technology & Genomics

O.J. Simpson capital murder case,1/95­9/95 Odds of blood in Ford Bronco not being R. Goldman’s: ■ 6.5 billion to 1 ■ Odds of blood on socks in bedroom not  being N. Brown­Simpson’s: ■ 8.5 billion to 1 Odds of blood on glove not being from R. Goldman, N. Brown­Simpson, 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 ■

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Recombinant DNA ■

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Def: DNA in which genes from 2 different sources are linked Genetic engineering: direct manipulation of genes for practical purposes Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products

Bacterial plasmids in gene cloning

DNA Cloning ■

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Restriction enzymes (endonucleases): in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific Restriction site: recognition sequence for a particular restriction enzyme Restriction fragments: segments of DNA cut by restriction enzymes in a reproducable way Sticky end: short extensions of restriction fragments DNA ligase: enzyme that can join the sticky ends of DNA fragments Cloning vector: DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)

Restriction Enzymes

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Steps for eukaryotic gene cloning ■

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Isolation of cloning vector (bacterial plasmid) & genesource DNA (gene of interest) Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase Introduction of cloning vector into cells (transformation by bacterial cells) Cloning of cells (and foreign genes) Identification of cell clones carrying the gene of interest

Cloning

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DNA Analysis & Genomics

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PCR (polymerase chain reaction) Gel electrophoresis Restriction fragment analysis (RFLPs) Southern blotting DNA sequencing

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Human genome project

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Polymerase chain reaction (PCR) ■

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Amplification of any piece of DNA without cells (in vitro) Materials: heat, DNA polymerase, nucleotides, singlestranded DNA primers Applications: fossils, forensics, prenatal diagnosis, etc.

DNA Analysis ■

Gel electrophoresis: separates nucleic acids or proteins on the basis of size or electrical charge creating DNA bands of the same length

Restriction fragment analysis ■ ■

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Restriction fragment length polymorphisms (RFLPs) Southern blotting: process that reveals sequences and the RFLPs in a DNA sequence DNA Fingerprinting

DNA Sequencing ■

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Determination of nucleotide sequences (Sanger method, sequencing machine) Genomics: the study of genomes based on DNA sequences Human Genome Project

Practical DNA Technology Uses ■ ■ ■

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Diagnosis of disease Human gene therapy Pharmaceutical products (vaccines) Forensics Animal husbandry (transgenic organisms) Genetic engineering in plants Ethical concerns?