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Benjamin A. Pierce GENETICS A Conceptual Approach FIFTH EDITION CHAPTER 19 Molecular Genetic Analysis and Biotechnology © 2014 W. H. Freeman and Company Techniques of Molecular Genetics Have Revolutionized Biology • Recombinant DNA Technology (Genetic Engineering) – Techniques for locating, isolating, altering, and studying DNA segments • The Molecular Genetics Revolution – Biotechnology: the use of these techniques to develop new products • Working at the Molecular Level Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes • First step: isolate DNA segment or gene from remaining DNA • Cutting and joining DNA fragments—restriction enzymes • Viewing DNA fragments • Locating DNA fragments with southern blotting and probes Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes • First step: isolate DNA segment or gene from remaining DNA • Cutting and joining DNA fragments—restriction enzymes • Viewing DNA fragments • Locating DNA fragments with southern blotting and probes Cutting and Joining DNA Fragments • Restriction enzymes: recognizing and cutting DNA at specific nucleotide sequences • Type II restriction enzyme: most useful enzyme • By adding methyl groups to the recognition sequence to protect itself from being digested by its own enzyme in bacteria Cutting and Joining DNA Fragments • Cohesive ends: fragments with short, singlestranded overhanging ends • Blunt ends: even-length ends from both single strands Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes • First step: isolate DNA segment or gene from remaining DNA • Cutting and joining DNA fragments—restriction enzymes • Viewing DNA Fragments • Locating DNA fragments with southern blotting and probes Viewing DNA Fragments • Gel electrophoresis • Autoradiography Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes • First step: isolate DNA segment or gene from remaining DNA • Cutting and joining DNA fragments—restriction enzymes • Viewing DNA fragments • Locating DNA fragments with southern blotting and probes Locating DNA Fragments with Southern Blotting and Probes • Probe: DNA or RNA with a base sequence complementary to a sequence in the gene of interest Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes • Cloning genes • Application: the genetic engineering of plants with pesticides • Amplifying DNA fragments with the polymerase chain reaction Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes • Cloning genes • Application: the genetic engineering of plants with pesticides • Amplifying DNA fragments with the polymerase chain reaction Cloning Genes • Gene cloning: amplifying a specific piece of DNA via a bacteria cell • Cloning vector: a replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell Cloning Genes • Plasmid vectors – Plasmids: circular DNA molecules from bacteria – Insert foreign DNA into plasmid using restriction enzymes – Linkers: synthetic DNA fragments containing restriction sites • Transformation of host cells with plasmids • Screening cells for recombinant plasmids – Selectable markers are used to confirm whether the cells have been transformed or not Cloning Genes • Other gene vectors: – – – – Cosmids Bacterial Artificial Chromosomes (BACs) Yeast Artificial Chromosome Ti plasmid Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes • Cloning genes • Application: the genetic engineering of plants with pesticides • Amplifying DNA fragments with the polymerase chain reaction Application: The Genetic Engineering of Plants with Pesticide • Bacillus thuringienis • Bt gene • Agrobacterium tumefaciens Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes • Cloning genes • Application: the genetic engineering of plants with pesticides • Amplifying DNA fragments with the polymerase chain reaction Amplifying DNA Fragments with the Polymerase Chain Reaction (PCR) • The PCR reaction – Taq polymerase: stable DNA polymerase at high temperature – Reverse-transcription PCR Amplifying DNA Fragments with the Polymerase Chain Reaction (PCR) • Limitations of PCR – Prior knowledge of target DNA – Contamination – Accuracy – Amplified fragments are less than 2 kb Amplifying DNA fragments with the polymerase chain reaction (PCR) • Applications of PCR – Real-time PCR: quantitatively determining the amount of DNA amplified as the reaction proceeds Molecular Techniques Can Be Used to Find Genes of Interest • Gene libraries • In situ hybridization • Positional cloning • Application: isolating the gene for cystic fibrosis Molecular Techniques Can Be Used to Find Genes of Interest • Gene libraries • In situ hybridization • Positional cloning • Application: isolating the gene for cystic fibrosis Gene Libraries • DNA library: a collection of clones containing all the DNA fragments from one source – Creating a genomic DNA library – cDNA libraries: consisting only of those DNA sequences that are transcribed into mRNA Gene Libraries • Screening DNA libraries – Plating clones of the library – Probing plated colonies or plaques Molecular Techniques Can Be Used to Find Genes of Interest • Gene libraries • In situ hybridization • Positional cloning • Application: isolating the gene for cystic fibrosis In Situ Hybridization • DNA probes used to determine the chromosomal location and to visualize a gene while it is in a cell • FISH Molecular Techniques Can Be Used to Find Genes of Interest • Gene libraries • In situ hybridization • Positional cloning • Application: isolating the gene for cystic fibrosis Positional Cloning • Isolating genes on the basis of their position on a genetic map • Chromosome walking • Chromosome jumping Molecular Techniques Can Be Used to Find Genes of Interest • Gene libraries • In situ hybridization • Positional cloning • Application: isolating the gene for cystic fibrosis Application: Isolating the Gene for Cystic Fibrosis • Autosomal recessive disorder • Characterized by chronic lung infections, insufficient pancreatic enzyme production, and increased salt concentration in sweat DNA Sequences Can Be Determined and Analyzed • Restriction Fragment Length Polymorphisms (RFLPs) • DNA sequencing • Next-generation sequencing technologies • DNA fingerprinting • Application: identifying people who died in the collapse of the World Trade Center DNA Sequences Can Be Determined and Analyzed • Restriction Fragment Length Polymorphisms (RFLPs) • DNA sequencing • Next-generation sequencing technologies • DNA fingerprinting • Application: identifying people who died in the collapse of the World Trade Center Restriction Fragment Length Polymorphisms • Some DNA fragments have different restriction sites due to mutation for the same restriction enzyme • Causes polymorphisms within a population DNA Sequences Can Be Determined and Analyzed • Restriction Fragment Length Polymorphisms (RFLPs) • DNA sequencing • Next-generation sequencing technologies • DNA fingerprinting • Application: identifying people who died in the collapse of the World Trade Center DNA Sequencing • Sanger’s dideoxy-sequencing method – Dideoxyribonucleoside triphosphate (ddNTP) lacks a 3′-oh group, which terminates DNA synthesis DNA Sequences Can Be Determined and Analyzed • Restriction Fragment Length Polymorphisms (RFLPs) • DNA sequencing • Next-generation sequencing technologies • DNA fingerprinting • Application: identifying people who died in the collapse of the World Trade Center Next-Generation Sequencing Technologies • Pyrosequencing • Illumina sequencing • Third-generation sequencing DNA Sequences Can Be Determined and Analyzed • Restriction Fragment Length Polymorphisms (RFLPs) • DNA sequencing • Next-generation sequencing technologies • DNA fingerprinting • Application: identifying people who died in the collapse of the World Trade Center DNA Fingerprinting (DNA Profiling) • Microsatellites: (short tandem repeats, STRs) variable number of copies of repeat sequences possessed by many organisms • Detected by PCR • Fragments represented as peaks on a graph – Homozygotes: single tall peak – Heterozygotes: two shorter peaks DNA Sequences Can Be Determined and Analyzed • Restriction Fragment Length Polymorphisms (RFLPs) • DNA sequencing • Next-generation sequencing technologies • DNA fingerprinting • Application: identifying people who died in the collapse of the World Trade Center Application: Identifying People Who Died in the Collapse of the World Trade Center • Usual means of victim ID were of little use with WTC remains • Used DNA fingerprinting • Also carried out on mitochondrial DNA • INSERT FIG 19.31 Molecular Techniques Are Increasingly Used to Analyze Gene Function • • • • • • • Forward and reverse genetics Creating random mutations Site-directed mutagenesis Transgenic animals Knockout mice Silencing genes with RNAi Application: Using RNAi for the treatment of human disease Forward and Reverse Genetics • Forward genetics: Begins with a phenotype to a gene that encodes the phenotype • Reverse genetics: Begins with a gene of unknown function, first inducing mutations and then checking the effect of the mutation on the phenotype Creating Random Mutations • A means to increase the number of mutants in an experimental population • Use mutagenic agents: radiation, chemical mutagens, transposable elements Site-Directed Mutagenesis • Site-directed mutagenesis • Oligonucleotide-directed mutagenesis Transgenic Animals • An organism permanently altered by the addition of a DNA sequence to its genome • Transgene Knockout Mice • A normal gene of the mouse has been fully disabled • Knock-in mice: a mouse carries an inserted DNA sequence at specific locations Silencing Genes with RNAi • siRNAs • Process called RNA interference (RNAi) Application: Using RNAi for the Treatment of Human Disease • High cholesterol • RNAi could be used to reduce the levels of ApoB and blood cholesterol in nonhuman primates Biotechnology Harnesses the Power of Molecular Genetics • Pharmaceutical products • Specialized bacteria • Agriculture products • Genetic testing • Gene therapy Concept Check 1 A geneticist is interested in the immune function of mice and induces random mutations in a number of genes in mice and then determines which of the resulting mutant mice have impaired immune function. This is an example of ________. a. b. c. d. forward genetics reverse genetics both forward and reverse genetics neither forward nor reverse genetics Concept Check 1 A geneticist is interested in the immune function of mice and induces random mutations in a number of genes in mice and then determines which of the resulting mutant mice have impaired immune function. This is an example of ________. a. b. c. d. forward genetics reverse genetics both forward and reverse genetics neither forward nor reverse genetics