Molecular Genetic Analysis and Biotechnology Benjamin A. Pierce GENETICS A Conceptual Approach FIFTH EDITION CHAPTER 19 Molecular Genetic Analysis and Biotechnology © 2014 W. H. Freeman and Company

Blindness affects 45 million people throughout the world Blindness affects 45 million people throughout the world. Genetic engineering is now being used to treat patients with Leber congenital amaurosis, a genetic form of blindness. [Paul Doyle/Alamy Images.]

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, single-stranded overhanging ends Blunt ends: even-length ends from both single strands

Figure 19.2a Restriction enzymes make double-stranded cuts in DNA, producing cohesive, or sticky, ends.

Figure 19.2b Restriction enzymes make double-stranded cuts in DNA, producing cohesive, or sticky, ends.

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

Figure 19.3 Gel electrophoresis can be used to separate DNA molecules on the basis of their size and electrical charge. [Photograph courtesy of Carol Eng.]

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

Figure 19.4 Southern blotting and hybridization with probes can locate a few specific fragments in a large pool of DNA.

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

Figure 19.5 An idealized cloning vector has an origin of replication, one or more selectable markers, and one or more unique restriction sites.

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

Figure 19. 6 The pUC19 plasmid is a typical cloning vector Figure 19.6 The pUC19 plasmid is a typical cloning vector. It contains a cluster of unique restriction sites, an origin of replication, and two selectable markers—an ampicillin-resistance gene and a lacZ gene.

Figure 19.7 A foreign DNA fragment can be inserted into a plasmid with the use of restriction enzymes.

Figure 19.8 The lacZ gene can be used to screen bacteria containing recombinant plasmids. A special plasmid carries a copy of the lacZ gene and an ampicillin-resistance gene. [Photograph: Cytographics/Visuals Unlimited.]

Cloning Genes Other gene vectors: Cosmids Bacterial Artificial Chromosomes (BACs) Yeast Artificial Chromosome Ti plasmid

Figure 19.9 To ensure transcription and translation, a foreign gene may be inserted into an expression vector—in this example, an E. coli expression vector.

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

Figure 19.11 The polymerase chain reaction is used to amplify even very small samples of DNA.

Figure 19.11 The polymerase chain reaction is used to amplify even very small samples of DNA.

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

Figure 19.14 A genomic library contains all of the DNA sequences found in an organism’s genome.

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

Figure 19.20 The gene for cystic fibrosis was located by positional cloning.

Figure 19.21 A candidate for the cystic fibrosis gene is expressed in pancreatic, respiratory, and sweat-gland tissues—tissues that are affected by the disease. Shown is a Northern blot of mRNA produced by the candidate gene in different tissues. These data provided evidence that the candidate gene is in fact the gene that causes cystic fibrosis. [After J. R. Riordan et al., Science 245:1066–1073, 1989.]

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

Figure 19.22 Restriction fragment length polymorphisms are genetic markers that can be used in mapping.

Figure 19.23 Restriction fragment length polymorphisms can be used to detect linkage. There is a close correspondence between the inheritance of the RFLP alleles and the presence of Huntington disease, indicating that the genes that encode the RFLP and Huntington disease are closely linked.

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

Figure 19.24 The dideoxy sequencing reaction requires a special substrate for DNA synthesis. (a) Structure of deoxyribonucleoside triphosphate, the normal substrate for DNA synthesis. (b) Structure of dideoxyribonucleoside triphosphate, which lacks an OH group on the 3′-carbon atom.

Figure 19.25 The dideoxy method of DNA sequencing is based on the termination of 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 Oligonucleotide-directed mutagenesis

Figure 19.32 Oligonucleotide-directed mutagenesis is used to study gene function when appropriate restriction sites are not available.

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