2DNA Cloning Plasmids -used to insert foreign DNA Recombinant plasmid is inserted into a bacteriaReproduction in bacteria cell results in cloning of the plasmid including foreign geneUseful for making copies of a gene and producing a protein product
3Making recombinant DNA Use bacterial restriction enzymesCuts DNA at specific restriction sitesCuts covalent bonds between sugar phosphate backboneMaking restriction fragmentsMost useful restriction enzymes cut DNA in a staggered way forming “sticky ends”DNA ligase seals the bonds between restriction fragments.Cloning vector is original plasmid carrying foreign gene into the host cell.
4Storing 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.A bacterial artificial chromosome (BAC)is a large plasmid that can carry a large insert with many genes.
5Storing Cloned Genes in DNA Libraries Complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell.cDNA library represents only part of the genome--only the subset of genes transcribed into mRNA in th original cells
6Reverse transcriptase Poly-A tail mRNA FigDNA in nucleusmRNAs in cytoplasmReverse transcriptasePoly-A tailmRNADNA strandPrimerDegraded mRNAFigure 20.6 Making complementary DNA (cDNA) for a eukaryotic gene
7Reverse transcriptase Poly-A tail mRNA FigDNA in nucleusmRNAs in cytoplasmReverse transcriptasePoly-A tailmRNADNA strandPrimerDegraded mRNAFigure 20.6 Making complementary DNA (cDNA) for a eukaryotic geneDNA polymerase
8Reverse transcriptase Poly-A tail mRNA FigDNA in nucleusmRNAs in cytoplasmReverse transcriptasePoly-A tailmRNADNA strandPrimerDegraded mRNAFigure 20.6 Making complementary DNA (cDNA) for a eukaryotic geneDNA polymerasecDNA
9Screening 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 geneThis process is called nucleic acid hybridization
10For example, if the desired gene is A probe can be synthesized that is complementary to the gene of interestFor example, if the desired gene is– Then we would synthesize this probe……5GGCTAACTTAGC33CCGATTGAATCG5
11The 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
12Radioactively labeled probe molecules Fig. 20-7TECHNIQUERadioactively labeled probe moleculesProbe DNAGene of interestMultiwell plates holding libraryclonesSingle-stranded DNA from cellFilm•Figure 20.7 Detecting a specific DNA sequence by hybridizing with a nucleic acid probeNylon membraneNylon membraneLocation of DNA with the complementary sequence
13Detecting DNA sequence by hybridization with a nucleic acid probe ResultsThe location of the black spot on the photographic film identifies the clone containing the gene of interest.By using probes with different nucleotide sequences, researchers can screen the collection of bacterial clones for different genes.
14Expressing Cloned Eukaryotic Genes After gene has been cloned, its protein products can be produced in larger amounts for research.Cloned genes can be expressed as protein in either bacterial or eukaryotic cells.
15Problems with expressing eukaryotic genes in bacterial host cells Scientists use an expression vector, a cloning vector that contains a highly active prokaryotic promoterThis allows the bacteria to recognize the promoter and proceed to express the foreign gene.This allows synthesis of many eukaryotic proteins in bacteria cells.Another problem is the presence of introns in eukaryotic genes.Bacteria cells do not have RNA splicing machinery.This can be overcome by using cDNA which includes only the exons.
16Eukaryotic cloning and Expression Systems The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problemsYACs behave normally in mitosis and can carry more DNA than a plasmidEukaryotic hosts can provide the post-translational modifications that many proteins require
17Amplifying DNA using Polymerase Chain reaction (PCR) PCR can produce many copies of a specific target segment of DNAThree-step cycle-heating--cooling--and replicationBrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
18molecules; 2 molecules (in white boxes) match target sequence Fig. 20-8TECHNIQUE53Target sequenceGenomic DNA351Denaturation53352AnnealingCycle 1 yields2moleculesPrimers3ExtensionNew nucleo- tidesFigure 20.8 The polymerase chain reaction (PCR)Cycle 2 yields4moleculesCycle 3 yields 8molecules; 2 molecules (in white boxes) match target sequence
19TECHNIQUE 5 3 Target sequence Genomic DNA 3 5 Fig. 20-8a Figure 20.8 The polymerase chain reaction (PCR)
22molecules; 2 molecules (in white boxes) match target sequence Fig. 20-8dCycle 3 yields 8molecules; 2 molecules (in white boxes) match target sequenceFigure 20.8 The polymerase chain reaction (PCR)
23Concept 20.2: DNA technology allows us to study the sequence, expression, and function of a gene DNA cloning allows researchers toCompare genes and alleles between individualsLocate gene expression in a bodyDetermine the role of a gene in an organismSeveral techniques are used to analyze the DNA of genes
24Gel Electrophoresis and Southern Blotting Gel electrophoresis uses a gel as a molecular sieve to separate nucleic acids by sizeA current is applied to the gel that causes the charged molecules to moveDNA is negatively charged and it moves towards a positive pole.Molecules are sorted into “bands” by their size
25Mixture of DNA mol- ecules of different sizes Fig. 20-9aTECHNIQUEPower sourceMixture of DNA mol- ecules of different sizes–CathodeAnode+Gel1Power sourceFigure 20.9 Gel electrophoresis–+Longer molecules2Shorter molecules
26Fig. 20-9bRESULTSFigure 20.9 Gel electrophoresis
27In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresisRestriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a geneThe procedure is also used to prepare pure samples of individual fragments
28FigNormal -globin alleleNormal alleleSickle-cell allele175 bp201 bpLarge fragmentDdeIDdeIDdeIDdeILarge fragmentSickle-cell mutant -globin allele376 bp201 bp 175 bp376 bpLarge fragmentDdeIFigure Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the β-globin geneDdeIDdeI(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles
29A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridizationSpecific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel
30Restriction fragments DNA + restriction enzyme I II III Fig aTECHNIQUEHeavy weightRestriction fragmentsDNA + restriction enzymeI II IIINitrocellulose membrane (blot)GelSpongeI Normal -globin alleleII Sickle-cell alleleIII HeterozygotePaper towelsAlkaline solutionFigure Southern blotting of DNA fragments1Preparation of restriction fragments2Gel electrophoresis3DNA transfer (blotting)
31Radioactively labeled probe for -globin gene Fig bRadioactively labeled probe for -globin geneProbe base-pairs with fragmentsI II IIII II IIIFragment from sickle-cell -globin alleleFilm over blotFigure Southern blotting of DNA fragmentsFragment from normal -globin alleleNitrocellulose blot4Hybridization with radioactive probe5Probe detection
32Studying the Expression of Interacting Groups of Genes Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assaysDNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions
33Labeled cDNA molecules (single strands) FigTECHNIQUETissue sample1Isolate mRNA.2Make cDNA by reverse transcription, using fluorescently labeled nucleotides.mRNA moleculesLabeled cDNA molecules (single strands)3Apply the cDNA mixture to a microarray, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray.DNA fragments representing specific genesFigure DNA microarray assay of gene expression levelsDNA microarrayDNA microarray with 2,400 human genes4Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample.
34Concept 20.3: Cloning organisms may lead to production of stem cells for research and other applicationsOrganismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell
35Cloning Plants: Single-Cell Cultures One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organismA totipotent cell is one that can generate a complete new organism
36EXPERIMENT RESULTS Transverse section of carrot root 2-mg fragments FigEXPERIMENTRESULTSTransverse section of carrot root2-mg fragmentsFigure Can a differentiated plant cell develop into a whole plant?Fragments were cultured in nu- trient medium; stirring caused single cells to shear off into the liquid.Single cells free in suspension began to divide.Embryonic plant developed from a cultured single cell.Plantlet was cultured on agar medium.Later it was planted in soil.A single somatic carrot cell developed into a mature carrot plant.
37Cloning Animals: Nuclear Transplantation In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cellExperiments with frog embryos have shown that a transplanted nucleus can often support normal development of the eggHowever, the older the donor nucleus, the lower the percentage of normally developing tadpoles
38EXPERIMENT RESULTS Frog embryo Frog egg cell Frog tadpole UV FigFrog embryoFrog egg cellFrog tadpoleEXPERIMENTUVFully differ-entiated(intestinal) cellLess differ- entiated cellDonor nucleus trans- plantedDonornucleustrans-plantedEnucleatedegg cellEgg with donor nucleusactivated to begindevelopmentRESULTSFigure Can the nucleus from a differentiated animal cell direct development of an organism?Most developinto tadpolesMost stop developingbefore tadpole stage
39Reproductive 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 cellDolly’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
40TECHNIQUE RESULTS Mammary cell donor Egg cell donor FigTECHNIQUEMammary cell donorEgg cell donor12Egg cell from ovaryNucleus removedCultured mammary cells3Cells fused3Nucleus from mammary cell4Grown in cultureEarly embryoFigure Reproductive cloning of a mammal by nuclear transplantationFor the Discovery Video Cloning, go to Animation and Video Files.5Implanted in uterus of a third sheepSurrogate mother6Embryonic developmentLamb (“Dolly”) genetically identical to mammary cell donorRESULTS
41Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogsCC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent”
42FigFigure CC, the first cloned cat, and her single parent
43Problems Associated with Animal Cloning In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birthMany 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
44Stem Cells of AnimalsA stem cell is a relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more typesStem cells isolated from early embryos at the blastocyst stage are called embryonic stem cells; these are able to differentiate into all cell typesThe adult body also has stem cells, which replace nonreproducing specialized cells
45From bone marrow in this example FigEmbryonic stem cellsAdult stem cellsEarly human embryo at blastocyst stage (mammalian equiva- lent of blastula)From bone marrow in this exampleCells generating all embryonic cell typesCells generating some cell typesCultured stem cellsDifferent culture conditionsFigure Working with stem cellsDifferent types of differentiated cellsLiver cellsNerve cellsBlood cells
46The aim of stem cell research is to supply cells for the repair of damaged or diseased organs
47Many fields benefit from DNA technology and genetic engineering Concept 20.4: The practical applications of DNA technology affect our lives in many waysMany fields benefit from DNA technology and genetic engineeringOne benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
48Human Gene TherapyGene therapy is the alteration of an afflicted individual’s genesGene therapy holds great potential for treating disorders traceable to a single defective geneVectors are used for delivery of genes into specific types of cells, for example bone marrowGene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations
49Insert RNA version of normal allele into retrovirus. FigCloned gene1Insert RNA version of normal allele into retrovirus.Viral RNA2Let retrovirus infect bone marrow cells that have been removed from the patient and cultured.Retrovirus capsid3Viral DNA carrying the normal allele inserts into chromosome.Bone marrow cell from patientFigure Gene therapy using a retroviral vectorBone marrow4Inject engineered cells into patient.
50Pharmaceutical Products Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases
51Protein Production in Cell Cultures Host cells in culture can be engineered to secrete a protein as it is madeThis is useful for the production of insulin, human growth hormones, and vaccines
52Environmental Cleanup Genetic engineering can be used to modify the metabolism of microorganismsSome modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materialsBiofuels make use of crops such as corn, soybeans, and cassava to replace fossil fuels
53Agricultural Applications DNA technology is being used to improve agricultural productivity and food quality
54Animal Husbandry Protein Production by “Pharm” Animals and Plants Genetic engineering of transgenic animals speeds up the selective breeding processTransgenic animals are made by introducing genes from one species into the genome of another animalBeneficial genes can be transferred between varieties or speciesProtein Production by “Pharm” Animals and PlantsTransgenic 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