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Chapter 20 : 

Chapter 20 DNA Technology and Genomics

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Overview: Understanding and Manipulating Genomes One of the greatest achievements of modern science Has been the sequencing of the human genome, which was largely completed by 2003 DNA sequencing accomplishments Have all depended on advances in DNA technology, starting with the invention of methods for making recombinant DNA

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DNA technology has launched a revolution in the area of biotechnology The manipulation of organisms or their genetic components to make useful products An example of DNA technology is the microarray A measurement of gene expression of thousands of different genes

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Concept 20.1: DNA cloning permits production of multiple copies of a specific gene or other DNA segment To work directly with specific genes Scientists have developed methods for preparing well-defined, gene-sized pieces of DNA in multiple identical copies, a process called gene cloning

DNA Cloning and Its Applications: A Preview : 

DNA Cloning and Its Applications: A Preview Most methods for cloning pieces of DNA in the laboratory Share certain general features, such as the use of bacteria and their plasmids

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Overview of gene cloning with a bacterial plasmid, showing various uses of cloned genes

Using Restriction Enzymes to Make Recombinant DNA : 

Using Restriction Enzymes to Make Recombinant DNA Bacterial restriction enzymes Cut DNA molecules at a limited number of specific DNA sequences, called restriction sites

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A restriction enzyme will usually make many cuts in a DNA molecule Yielding a set of restriction fragments The most useful restriction enzymes cut DNA in a staggered way Producing fragments with “sticky ends” that can bond with complementary “sticky ends” of other fragments DNA ligase is an enzyme That seals the bonds between restriction fragments

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Using a restriction enzyme and DNA ligase to make recombinant DNA

Cloning a Eukraryotic Gene in a Bacterial Plasmid : 

Cloning a Eukraryotic Gene in a Bacterial Plasmid In gene cloning, the original plasmid is called a cloning vector Defined as a DNA molecule that can carry foreign DNA into a cell and replicate there

Producing Clones of Cells : 

Producing Clones of Cells

Identifying Clones Carrying a Gene of Interest : 

Identifying Clones Carrying a Gene of Interest A clone carrying the gene of interest Can be identified with a radioactively labeled nucleic acid probe that has a sequence complementary to the gene, a process called nucleic acid hybridization

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Nucleic acid probe hybridization

Storing Cloned Genes in DNA Libraries : 

Storing Cloned Genes in DNA Libraries A genomic library made using bacteria Is the collection of recombinant vector clones produced by cloning DNA fragments derived from an entire genome

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A genomic library made using bacteriophages Is stored as a collection of phage clones

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A complementary DNA (cDNA) library Is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell

Cloning and Expressing Eukaryotic Genes : 

Cloning and Expressing Eukaryotic Genes As an alternative to screening a DNA library for a particular nucleotide sequence The clones can sometimes be screened for a desired gene based on detection of its encoded protein

Bacterial Expression Systems : 

Bacterial Expression Systems Several technical difficulties Hinder the expression of cloned eukaryotic genes in bacterial host cells To overcome differences in promoters and other DNA control sequences Scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoter

Eukaryotic Cloning and Expression Systems : 

Eukaryotic Cloning and Expression Systems The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors Helps avoid gene expression problems

Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) : 

Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) The polymerase chain reaction, PCR Can produce many copies of a specific target segment of DNA Uses primers that bracket the desired sequence Uses a heat-resistant DNA polymerase

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The PCR procedure

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Concept 20.2: Restriction fragment analysis detects DNA differences that affect restriction sites Restriction fragment analysis Can rapidly provide useful comparative information about DNA sequences

Gel Electrophoresis and Southern Blotting : 

Gel Electrophoresis and Southern Blotting Gel electrophoresis Separates DNA restriction fragments of different lengths

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Restriction fragment analysis Is useful for comparing two different DNA molecules, such as two alleles for a gene

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Specific DNA fragments can be identified by Southern blotting Using labeled probes that hybridize to the DNA immobilized on a “blot” of the gel

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Southern blotting of DNA fragments

Restriction Fragment Length Differences as Genetic Markers : 

Restriction Fragment Length Differences as Genetic Markers Restriction fragment length polymorphisms (RFLPs) Are differences in DNA sequences on homologous chromosomes that result in restriction fragments of different lengths

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Specific fragments Can be detected and analyzed by Southern blotting The thousands of RFLPs present throughout eukaryotic DNA Can serve as genetic markers

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Concept 20.3: Entire genomes can be mapped at the DNA level The Human Genome Project Sequenced the human genome Scientists have also sequenced genomes of other organisms Providing important insights of general biological significance

Genetic (Linkage) Mapping: Relative Ordering of Markers : 

Genetic (Linkage) Mapping: Relative Ordering of Markers The initial stage in mapping a large genome Is to construct a linkage map of several thousand genetic markers spaced throughout each of the chromosomes

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The order of the markers and the relative distances between them on such a map Are based on recombination frequencies

Physical Mapping: Ordering DNA Fragments : 

Physical Mapping: Ordering DNA Fragments A physical map Is constructed by cutting a DNA molecule into many short fragments and arranging them in order by identifying overlaps Gives the actual distance in base pairs between markers

DNA Sequencing : 

DNA Sequencing Relatively short DNA fragments Can be sequenced by the dideoxy chain-termination method

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Dideoxy chain-termination method for sequencing DNA

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Linkage mapping, physical mapping, and DNA sequencing Represent the overarching strategy of the Human Genome Project An alternative approach to sequencing whole genomes starts with the sequencing of random DNA fragments Powerful computer programs would then assemble the resulting very large number of overlapping short sequences into a single continuous sequence

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Concept 20.4: Genome sequences provide clues to important biological questions In genomics Scientists study whole sets of genes and their interactions

Identifying Protein-Coding Genes in DNA Sequences : 

Identifying Protein-Coding Genes in DNA Sequences Computer analysis of genome sequences Helps researchers identify sequences that are likely to encode proteins

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Current estimates are that the human genome contains about 25,000 genes But the number of human proteins is much larger

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Comparison of the sequences of “new” genes With those of known genes in other species may help identify new genes

Determining Gene Function : 

Determining Gene Function For a gene of unknown function Experimental inactivation of the gene and observation of the resulting phenotypic effects can provide clues to its function

Studying Expression of Interacting Groups of Genes : 

Studying Expression of Interacting Groups of Genes DNA microarray assays allow researchers to compare patterns of gene expression In different tissues, at different times, or under different conditions

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DNA microarray assay of gene expression levels

Comparing Genomes of Different Species : 

Comparing Genomes of Different Species Comparative studies of genomes from related and widely divergent species Are providing valuable information in many fields of biology

Future Directions in Genomics : 

Future Directions in Genomics Genomics Is the study of entire genomes Proteomics Is the systematic study of all the proteins encoded by a genome Single nucleotide polymorphisms (SNPs) Provide useful markers for studying human genetic variation

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Concept 20.5: The practical applications of DNA technology affect our lives in many ways Numerous fields are benefiting from DNA technology and genetic engineering

Medical Applications : 

Medical Applications One obvious benefit of DNA technology Is the identification of human genes whose mutation plays a role in genetic diseases

Diagnosis of Diseases : 

Diagnosis of Diseases Medical scientists can now diagnose hundreds of human genetic disorders By using PCR and primers corresponding to cloned disease genes, then sequencing the amplified product to look for the disease-causing mutation

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Even when a disease gene has not yet been cloned The presence of an abnormal allele can be diagnosed with reasonable accuracy if a closely linked RFLP marker has been found

Human Gene Therapy : 

Human Gene Therapy Gene therapy Is the alteration of an afflicted individual’s genes Holds great potential for treating disorders traceable to a single defective gene Uses various vectors for delivery of genes into cells

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Gene therapy using a retroviral vector

Pharmaceutical Products : 

Pharmaceutical Products Applications of DNA technology include Large-scale production of human hormones and other proteins with therapeutic uses Production of safer vaccines

Forensic Evidence : 

Forensic Evidence DNA “fingerprints” obtained by analysis of tissue or body fluids found at crime scenes Can provide definitive evidence that a suspect is guilty or not

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A DNA fingerprint Is a specific pattern of bands of RFLP markers on a gel Figure 20.17

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DNA fingerprinting Can also be used in establishing paternity

Environmental Cleanup : 

Environmental Cleanup Genetic engineering can be used to modify the metabolism of microorganisms So that they can be used to extract minerals from the environment or degrade various types of potentially toxic waste materials

Agricultural Applications : 

Agricultural Applications DNA technology Is being used to improve agricultural productivity and food quality

Animal Husbandry and “Pharm” Animals : 

Animal Husbandry and “Pharm” Animals Transgenic animals Contain genes from other organisms

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Have been engineered to be pharmaceutical “factories”

Genetic Engineering in Plants : 

Genetic Engineering in Plants Agricultural scientists Have already endowed a number of crop plants with genes for desirable traits

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The Ti plasmid Is the most commonly used vector for introducing new genes into plant cells

Safety and Ethical Questions Raised by DNA Technology : 

Safety and Ethical Questions Raised by DNA Technology The potential benefits of genetic engineering Must be carefully weighed against the potential hazards of creating products or developing procedures that are harmful to humans or the environment

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Today, most public concern about possible hazards Centers on genetically modified (GM) organisms used as food