Biotechnology: Unit 3 - PDF

Unit 3 Biotechnology PowerPoint® Lecture Presentations for Biology Eighth Edition Neil Campbell and Jane Reece Lectures by Chris Romero, updated by Erin Barley with contributions from Joan Sharp Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings O...

Unit 3 Biotechnology PowerPoint® Lecture Presentations for Biology Eighth Edition Neil Campbell and Jane Reece Lectures by Chris Romero, updated by Erin Barley with contributions from Joan Sharp Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings Overview: The DNA Toolbox Sequencing of the human genome was completed by 2007 DNA sequencing has depended on advances in technology, starting with making recombinant DNA In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings  Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes DNA technology has revolutionized 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 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-1 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-1 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-1 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-1  https://tinyurl.com/LabXChange-DNAExtraction Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Concept 20.1: DNA cloning yields multiple copies of a gene or other DNA segment To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called DNA cloning Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings DNA Cloning and Its Applications: A Preview Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome Cloned genes are useful for making copies of a particular gene and producing a protein product Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings  Gene cloning involves using bacteria to make multiple copies of a gene Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA This results in the production of multiple copies of a single gene Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-2 Cell containing gene Bacterium of interest 1 Gene inserted into plasmid Bacterial Plasmid chromosome Gene of Recombinant interest DNA of DNA (plasmid) chromosome 2 Plasmid put into bacterial cell Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Protein expressed Interest by gene of interest Copies of gene Protein harvested 4 Basic research and Basic various applications Basic research research on gene on protein Gene for pest Gene used to alter Protein dissolves Human growth hor- resistance inserted bacteria for cleaning blood clots in heart mone treats stunted into plants up toxic waste attack therapy growth Fig. 20-2a Cell containing gene Bacterium of interest 1 Gene inserted into plasmid Bacterial Plasmid chromosome Gene of Recombinant interest DNA of DNA (plasmid) 2 chromosome 2 Plasmid put into bacterial cell Recombinant bacterium Fig. 20-2b Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Protein expressed Interest by gene of interest Copies of gene Protein harvested 4 Basic research and Basic various applications Basic research research on gene on protein Gene for pest Gene used to alter Protein dissolves Human growth hor- resistance inserted bacteria for cleaning blood clots in heart mone treats stunted into plants up toxic waste attack therapy growth Using Restriction Enzymes to Make Recombinant DNA Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites A restriction enzyme usually makes many cuts, yielding restriction fragments The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other fragments Animation: Restriction Enzymes Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings  DNA ligase is an enzyme that seals the bonds between restriction fragments Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-3-1 Restriction site DNA 5 3 3 5 1 Restriction enzyme cuts sugar-phosphate backbones. Sticky end Fig. 20-3-2 Restriction site DNA 5 3 3 5 1 Restriction enzyme cuts sugar-phosphate backbones. Sticky end 2 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. One possible combination Fig. 20-3-3 Restriction site DNA 5 3 3 5 1 Restriction enzyme cuts sugar-phosphate backbones. Sticky end 2 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. One possible combination 3 DNA ligase seals strands. Recombinant DNA molecule Cloning a Eukaryotic Gene in a Bacterial Plasmid In gene cloning, the original plasmid is called a cloning vector A cloning vector is a DNA molecule that can carry foreign DNA into a host cell and replicate there Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Producing Clones of Cells Carrying Recombinant Plasmids Several steps are required to clone the hummingbird β-globin gene in a bacterial plasmid: – The hummingbird genomic DNA and a bacterial plasmid are isolated – Both are digested with the same restriction enzyme – The fragments are mixed, and DNA ligase is added to bond the fragment sticky ends Animation: Cloning a Gene Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings  – Some recombinant plasmids now contain hummingbird DNA – The DNA mixture is added to bacteria that have been genetically engineered to accept it – The bacteria are plated on a type of agar that selects for the bacteria with recombinant plasmids – This results in the cloning of many hummingbird DNA fragments, including the β-globin gene Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-4-1 TECHNIQUE Hummingbird cell Bacterial cell lacZ gene Restriction Sticky Gene of interest site ends ampR gene Bacterial Hummingbird plasmid DNA fragments Fig. 20-4-2 TECHNIQUE Hummingbird cell Bacterial cell lacZ gene Restriction Sticky Gene of interest site ends ampR gene Bacterial Hummingbird plasmid DNA fragments Nonrecombinant plasmid Recombinant plasmids Fig. 20-4-3 TECHNIQUE Hummingbird cell Bacterial cell lacZ gene Restriction Sticky Gene of interest site ends ampR gene Bacterial Hummingbird plasmid DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carrying plasmids Fig. 20-4-4 TECHNIQUE Hummingbird cell Bacterial cell lacZ gene Restriction Sticky Gene of interest site ends ampR gene Bacterial Hummingbird plasmid DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carrying plasmids RESULTS Colony carrying non- Colony carrying recombinant recombinant plasmid plasmid with disrupted lacZ gene with intact lacZ gene One of many bacterial clones Storing Cloned Genes in DNA Libraries A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome A genomic library that is made using bacteriophages is stored as a collection of phage clones Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-5 Foreign genome Large insert cut up with Large plasmid with many genes restriction enzyme or BAC Recombinant clone phage DNA Bacterial Recombinant clones plasmids Phage clones (a) Plasmid library (b) Phage library (c) A library of bacterial artificial chromosome (BAC) clones Fig. 20-5a Foreign genome cut up with restriction enzyme or Recombinant phage DNA Bacterial Recombinant clones plasmids Phage clones (a) Plasmid library (b) Phage library  A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert BACs are another type of vector used in DNA library construction Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-5b Large insert Large plasmid with many genes BAC clone (c) A library of bacterial artificial chromosome (BAC) clones  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 A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-6-1 DNA in nucleus mRNAs in cytoplasm Fig. 20-6-2 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA DNA Primer strand Fig. 20-6-3 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA DNA Primer Degraded strand mRNA Fig. 20-6-4 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA DNA Primer Degraded strand mRNA DNA polymerase Fig. 20-6-5 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA DNA Primer Degraded strand mRNA DNA polymerase cDNA Screening a Library for Clones Carrying a Gene of Interest A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene This process is called nucleic acid hybridization Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings  A probe can be synthesized that is complementary to the gene of interest For example, if the desired gene is 5 … G G C T AA C TT A G C … 3 – Then we would synthesize this probe 3 C C G A TT G A A T C G 5 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings  The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest Once identified, the clone carrying the gene of interest can be cultured Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-7 TECHNIQUE Radioactively labeled probe molecules Probe DNA Gene of Multiwell plates interest holding library Single-stranded Film clones DNA from cell Nylon membrane Nylon Location of membrane DNA with the complementary sequence Expressing Cloned Eukaryotic Genes After a gene has been cloned, its protein product can be produced in larger amounts for research Cloned genes can be expressed as protein in either bacterial or eukaryotic cells Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Bacterial Expression Systems Several technical difficulties hinder 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 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 YACs behave normally in mitosis and can carry more DNA than a plasmid Eukaryotic hosts can provide the post- translational modifications that many proteins require Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings  One method of introducing recombinant DNA into eukaryotic cells is electroporation, applying a brief electrical pulse to create temporary holes in plasma membranes Alternatively, scientists can inject DNA into cells using microscopically thin needles Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-8 5 3 TECHNIQUE Target sequence Genomic DNA 3 5 1 Denaturation 5 3 3 5 2 Annealing Cycle 1 yields Primers 2 molecules 3 Extension New nucleo- tides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Fig. 20-8a 5 3 TECHNIQUE Target sequence Genomic DNA 3 5 Fig. 20-8b 1 Denaturation 5 3 3 5 2 Annealing Cycle 1 yields Primers 2 molecules 3 Extension New nucleo- tides Fig. 20-8c Cycle 2 yields 4 molecules Fig. 20-8d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence https://tinyurl.com/LabXChange-PCR Concept 20.2: DNA technology allows us to study the sequence, expression, and function of a gene DNA cloning allows researchers to – Compare genes and alleles between individuals – Locate gene expression in a body – Determine the role of a gene in an organism Several techniques are used to analyze the DNA of genes Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Gel Electrophoresis and Southern Blotting One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size A current is applied that causes charged molecules to move through the gel Molecules are sorted into “bands” by their size Video: Biotechnology Lab Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-9 TECHNIQUE Mixture of Power DNA mol- source ecules of – Cathode Anode + different sizes Gel 1 Power source – + Longer molecules 2 Shorter molecules RESULTS Fig. 20-9a TECHNIQUE Mixture of Power DNA mol- source ecules of – Cathode Anode + different sizes Gel 1 Power source – + Longer molecules 2 Shorter molecules Fig. 20-9b RESULTS  In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene The procedure is also used to prepare pure samples of individual fragments Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-10 Normal -globin allele Normal Sickle-cell allele allele 175 bp 201 bp Large fragment DdeI DdeI DdeI DdeI Large fragment Sickle-cell mutant -globin allele 376 bp 376 bp Large fragment 201 bp 175 bp DdeI DdeI DdeI (a) DdeI restriction sites in normal and (b) Electrophoresis of restriction fragments sickle-cell alleles of -globin gene from normal and sickle-cell alleles  A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-11 TECHNIQUE Heavy Restriction I II III weight DNA + restriction enzyme Nitrocellulose fragments membrane (blot) Gel Sponge I Normal II Sickle-cell III Heterozygote Paper -globin allele Alkaline towels allele solution 1 Preparation of restriction fragments 2 Gel electrophoresis 3 DNA transfer (blotting) Radioactively labeled probe for -globin gene Probe base-pairs I II III with fragments I II III Fragment from sickle-cell -globin allele Film over Fragment from blot normal -globin Nitrocellulose blot allele 4 Hybridization with radioactive probe 5 Probe detection Fig. 20-11a TECHNIQUE Heavy Restriction I II III weight DNA + restriction enzyme Nitrocellulose fragments membrane (blot) Gel Sponge I Normal II Sickle-cell III Heterozygote Paper -globin allele Alkaline solution towels allele 1 Preparation of restriction fragments 2 Gel electrophoresis 3 DNA transfer (blotting) Fig. 20-11b Radioactively labeled probe for -globin gene Probe base-pairs I II III with fragments I II III Fragment from sickle-cell -globin allele Film over Fragment from blot normal -globin Nitrocellulose blot allele 4 Hybridization with radioactive probe 5 Probe detection https://tinyurl.com/LabXChange- GelElectrophoresis DNA Sequencing Relatively short DNA fragments can be sequenced by the dideoxy chain termination method Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment The DNA sequence can be read from the resulting spectrogram Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-12 TECHNIQUE DNA Primer Deoxyribonucleotides Dideoxyribonucleotides (template strand) (fluorescently tagged) dATP ddATP dCTP ddCTP dTTP ddTTP DNA polymerase dGTP ddGTP DNA (template Labeled strands strand) Shortest Longest Direction of movement Longest labeled strand of strands Detector Laser Shortest labeled strand RESULTS Last base of longest labeled strand Last base of shortest labeled strand Fig. 20-12a TECHNIQUE DNA Primer Deoxyribonucleotides Dideoxyribonucleotides (template strand) (fluorescently tagged) dATP ddATP dCTP ddCTP DNA dTTP ddTTP polymerase dGTP ddGTP Fig. 20-12b TECHNIQUE DNA (template Labeled strands strand) Shortest Longest Direction of movement Longest labeled strand of strands Detector Laser Shortest labeled strand RESULTS Last base of longest labeled strand Last base of shortest labeled strand Analyzing Gene Expression Nucleic acid probes can hybridize with mRNAs transcribed from a gene Probes can be used to identify where or when a gene is transcribed in an organism Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Studying the Expression of Single Genes Changes in the expression of a gene during embryonic development can be tested using – Northern blotting – Reverse transcriptase-polymerase chain reaction Both methods are used to compare mRNA from different developmental stages Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings  Northern blotting combines gel electrophoresis of mRNA followed by hybridization with a probe on a membrane Identification of mRNA at a particular developmental stage suggests protein function at that stage Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings  Reverse transcriptase-polymerase chain reaction (RT-PCR) is quicker and more sensitive Reverse transcriptase is added to mRNA to make cDNA, which serves as a template for PCR amplification of the gene of interest The products are run on a gel and the mRNA of interest identified Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-13 TECHNIQUE 1 cDNA synthesis mRNAs cDNAs Primers 2 PCR amplification -globin gene 3 Gel electrophoresis RESULTS Embryonic stages 1 2 3 4 5 6  In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-14 50 µm Studying the Expression of Interacting Groups of Genes Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-15 TECHNIQUE Tissue sample 1 Isolate mRNA. 2 Make cDNA by reverse mRNA molecules transcription, using fluorescently labeled nucleotides. Labeled cDNA molecules (single strands) 3 Apply the cDNA mixture to a DNA fragments microarray, a different gene in representing each spot. The cDNA hybridizes specific genes with any complementary DNA on the microarray. DNA microarray DNA microarray 4 Rinse off excess cDNA; scan with 2,400 microarray for fluorescence. human genes Each fluorescent spot represents a gene expressed in the tissue sample. Determining Gene Function One way to determine function is to disable the gene and observe the consequences Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its function When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings  Gene expression can also be silenced using RNA interference (RNAi) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the gene’s mRNA Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Concept 20.3: Cloning organisms may lead to production of stem cells for research and other applications Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Cloning Plants: Single-Cell Cultures One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organism A totipotent cell is one that can generate a complete new organism Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-16 EXPERIMENT RESULTS Transverse section of carrot root 2-mg fragments Fragments were Single Embryonic Plantlet was A single cultured in nu- cells plant developed cultured on somatic trient medium; free in from a cultured agar medium. carrot cell stirring caused suspension single cell. Later it was developed single cells to began to planted into a mature shear off into divide. in soil. carrot plant. the liquid. Cloning Animals: Nuclear Transplantation In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell Experiments with frog embryos have shown that a transplanted nucleus can often support normal development of the egg However, the older the donor nucleus, the lower the percentage of normally developing tadpoles Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-17 EXPERIMENT Frog embryo Frog egg cell Frog tadpole UV Fully differ- Less differ- entiated entiated cell (intestinal) cell Donor Donor nucleus Enucleated nucleus trans- egg cell trans- planted planted Egg with donor nucleus activated to begin development RESULTS Most develop Most stop developing into tadpoles before tadpole stage Reproductive Cloning of Mammals In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell Dolly’s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-18 TECHNIQUE Mammary Egg cell cell donor donor 1 2 Egg cell from ovary Nucleus removed Cultured 3 Cells fused mammary cells 3 Nucleus from mammary cell 4 Grown in culture Early embryo 5 Implanted in uterus of a third sheep Surrogate mother 6 Embryonic development Lamb (“Dolly”) RESULTS genetically identical to mammary cell donor  Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent” Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-19 Problems Associated with Animal Cloning In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth Many epigenetic changes, such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Stem Cells of Animals A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types Stem cells isolated from early embryos at the blastocyst stage are called embryonic stem cells; these are able to differentiate into all cell types The adult body also has stem cells, which replace nonreproducing specialized cells Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-20 Embryonic stem cells Adult stem cells Early human embryo From bone marrow at blastocyst stage in this example (mammalian equiva- lent of blastula) Cells generating Cells generating all embryonic some cell types cell types Cultured stem cells Different culture conditions Different Liver cells Nerve cells Blood cells types of differentiated cells  The aim of stem cell research is to supply cells for the repair of damaged or diseased organs Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Concept 20.4: The practical applications of DNA technology affect our lives in many ways Many fields benefit from DNA technology and genetic engineering Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Medical Applications One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Diagnosis of Diseases Scientists can diagnose many 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 Genetic disorders can also be tested for using genetic markers that are linked to the disease- causing allele Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings  Single nucleotide polymorphisms (SNPs) are useful genetic markers These are single base-pair sites that vary in a population When a restriction enzyme is added, SNPs result in DNA fragments with different lengths, or restriction fragment length polymorphism (RFLP) Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-21 DNA T Normal allele SNP C Disease-causing allele Human Gene Therapy Gene therapy is the alteration of an afflicted individual’s genes Gene therapy holds great potential for treating disorders traceable to a single defective gene Vectors are used for delivery of genes into specific types of cells, for example bone marrow Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-22 Cloned gene 1 Insert RNA version of normal allele into retrovirus. Viral RNA 2 Let retrovirus infect bone marrow cells Retrovirus that have been removed from the capsid patient and cultured. 3 Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient 4 Inject engineered Bone cells into patient. marrow Pharmaceutical Products Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Synthesis of Small Molecules for Use as Drugs The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia- causing receptor Pharmaceutical products that are proteins can be synthesized on a large scale Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Protein Production in Cell Cultures Host cells in culture can be engineered to secrete a protein as it is made This is useful for the production of insulin, human growth hormones, and vaccines Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Protein Production by “Pharm” Animals and Plants Transgenic animals are made by introducing genes from one species into the genome of another animal Transgenic animals are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use “Pharm” plants are also being developed to make human proteins for medical use Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-23 Fig. 20-23a Fig. 20-23b Forensic Evidence and Genetic Profiles An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids Genetic profiles can be used to provide evidence in criminal and paternity cases and to identify human remains Genetic profiles can be analyzed using RFLP analysis by Southern blotting Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings  Even more sensitive is the use of genetic markers called short tandem repeats (STRs), which are variations in the number of repeats of specific DNA sequences PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths The probability that two people who are not identical twins have the same STR markers is exceptionally small Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-24 (a) This photo shows Earl Washington just before his release in 2001, after 17 years in prison. Source of STR STR STR sample marker 1 marker 2 marker 3 Semen on victim 17, 19 13, 16 12, 12 Earl Washington 16, 18 14, 15 11, 12 Kenneth Tinsley 17, 19 13, 16 12, 12 (b) These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder. Environmental Cleanup Genetic engineering can be used to modify the metabolism of microorganisms Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials Biofuels make use of crops such as corn, soybeans, and cassava to replace fossil fuels Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Agricultural Applications DNA technology is being used to improve agricultural productivity and food quality Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Animal Husbandry Genetic engineering of transgenic animals speeds up the selective breeding process Beneficial genes can be transferred between varieties or species Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Genetic Engineering in Plants Agricultural scientists have endowed a number of crop plants with genes for desirable traits The Ti plasmid is the most commonly used vector for introducing new genes into plant cells Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance, increased resistance to pests, increased resistance to salinity, and improved nutritional value of crops Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-25 TECHNIQUE Agrobacterium tumefaciens Ti plasmid Site where restriction enzyme cuts T DNA DNA with RESULTS the gene of interest Recombinant Ti plasmid Plant with new trait Safety and Ethical Questions Raised by DNA Technology Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings  Most public concern about possible hazards centers on genetically modified (GM) organisms used as food Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings  As biotechnology continues to change, so does its use in agriculture, industry, and medicine National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-UN3 DNA fragments from genomic DNA Vector or cDNA or copy of DNA obtained by PCR Cut by same restriction enzyme, mixed, and ligated Recombinant DNA plasmids Fig. 20-UN4 5 TCCATGAATTCTAAAGCGCTTATGAATTCACGGC 3 3 AGGTACTTAAGATTTCGCGAATACTTAAGTGCCG 5 Aardvark DNA A Plasmid Fig. 20-UN5 Fig. 20-UN6 Fig. 20-UN7 You should now be able to: 1. Describe the natural function of restriction enzymes and explain how they are used in recombinant DNA technology 2. Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid 3. Define and distinguish between genomic libraries using plasmids, phages, and cDNA 4. Describe the polymerase chain reaction (PCR) and explain the advantages and limitations of this procedure Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 5. Explain how gel electrophoresis is used to analyze nucleic acids and to distinguish between two alleles of a gene 6. Describe and distinguish between the Southern blotting procedure, Northern blotting procedure, and RT-PCR 7. Distinguish between gene cloning, cell cloning, and organismal cloning 8. Describe how nuclear transplantation was used to produce Dolly, the first cloned sheep Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9. Describe the application of DNA technology to the diagnosis of genetic disease, the development of gene therapy, vaccine production, and the development of pharmaceutical products 10.Define a SNP and explain how it may produce a RFLP 11.Explain how DNA technology is used in the forensic sciences Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 12.Discuss the safety and ethical questions related to recombinant DNA studies and the biotechnology industry Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings