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Overview: The DNA Toolbox • Sequencing of the human genome was completed by 2007 • Genetic engineering, the direct manipulation of genes for practical purposes • 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 • DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products 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 • Gene cloning produces multiple copies of gene-sized pieces of DNA. • Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings DNA Cloning and Its Applications: A Preview • 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 Fig. 20-2 Cell containing gene of interest Bacterium 1 Gene inserted into plasmid Bacterial Plasmid chromosome Recombinant DNA (plasmid) Gene of interest DNA of 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 Interest Protein expressed by gene of interest Copies of gene Basic Protein harvested 4 Basic research and various applications research on gene Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Basic research on protein Human growth hormone treats stunted growth DNA Cloning and Its Applications: A Preview • Gene cloning is useful for two basic purposes: • To make many copies of a particular gene. Isolated copies of a cloned gene may enable scientists to determine the gene’s nucleotide sequence or provide an organism with a new metabolic capability, such as pest resistance. • To create a protein product. Alternatively, a protein with medical uses, such as human growth hormone, can be harvested in large quantities from cultures of bacteria carrying the cloned gene for the protein. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-3-1 Restriction site DNA 1 5 3 3 5 Restriction enzyme cuts sugar-phosphate backbones. Sticky end Fig. 20-3-2 Restriction site DNA 1 5 3 3 5 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 • DNA ligase is an enzyme that seals the bonds between restriction fragments Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-3-3 Restriction site DNA 1 5 3 3 5 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 • 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 Fig. 20-4-4 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restriction site ampR gene Bacterial plasmid Sticky ends Gene of interest Hummingbird DNA fragments “Shotgun Approach” Nonrecombinant plasmid Thousand’s of Recombinant plasmids Bacteria carrying plasmids RESULTS Colony carrying nonrecombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted 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 • 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. 20-5 Foreign genome cut up with restriction enzyme Large insert Large plasmid with many genes or BAC clone Recombinant phage DNA Bacterial clones (a) Plasmid library Recombinant plasmids (b) Phage library Phage clones (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. 20-6-5 DNA in nucleus mRNAs in cytoplasm mRNA Reverse transcriptase Poly-A tail DNA Primer strand Degraded mRNA DNA polymerase cDNA Screening a Library for Clones Carrying a Gene of Interest (Researching the library) • 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 Multiwell plates holding library clones Probe DNA Gene of interest Single-stranded DNA from cell Film • Nylon membrane Nylon Location of membrane DNA with the complementary sequence Expressing Cloned Eukaryotic Genes (reading the book…) • 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 posttranslational 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 TECHNIQUE 3 Target sequence 3 Genomic DNA 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension New nucleotides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence http://www.sumanasinc.com/webcontent/anim ations/content/pcr.html 1 Denaturation 5 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 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-9 TECHNIQUE Mixture of DNA molecules of different sizes Power source – Cathode Anode + Gel 1 Power source – + Longer molecules 2 RESULTS Shorter molecules Fig. 20-9a TECHNIQUE Mixture of DNA molecules of different sizes Power source – Cathode Anode + Gel 1 Power source – + Longer molecules 2 Shorter molecules • 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 175 bp DdeI Sickle-cell allele Large fragment 201 bp DdeI Normal allele DdeI DdeI Large fragment Sickle-cell mutant -globin allele 376 bp DdeI 201 bp 175 bp Large fragment 376 bp DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene (b) Electrophoresis of restriction fragments 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 DNA + restriction enzyme Restriction fragments I II III Heavy weight Nitrocellulose membrane (blot) Gel Sponge I Normal II Sickle-cell III Heterozygote -globin allele allele 2 Gel electrophoresis 1 Preparation of restriction fragments Paper towels Alkaline solution 3 DNA transfer (blotting) Radioactively labeled probe for -globin gene I II III Probe base-pairs with fragments Fragment from sickle-cell -globin allele Nitrocellulose blot Fragment from normal -globin allele 4 Hybridization with radioactive probe I II III Film over blot 5 Probe detection Fig. 20-11b Radioactively labeled probe for -globin gene I II III Probe base-pairs with fragments Fragment from sickle-cell -globin allele Fragment from normal -globin Nitrocellulose blot allele 4 Hybridization with radioactive probe I II III Film over blot 5 Probe detection • 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 Sequencing and Analyzing Gene Expression • Dideoxyribonuclotide (dideoxy) chain termination method is an automated process that sequences the gene. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Studying the Expression of Single Genes • 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 • 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 Fig. 20-14 In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism 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 1 Isolate mRNA. 2 Make cDNA by reverse transcription, using fluorescently labeled nucleotides. 3 Apply the cDNA mixture to a microarray, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. Tissue sample mRNA molecules Labeled cDNA molecules (single strands) DNA fragments representing specific genes DNA microarray 4 Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample. DNA microarray with 2,400 human genes 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 • 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 • In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell • The current interest in organismal cloning arises primarily from it potential to generate stem cells. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 cell donor Egg cell donor 2 1 Egg cell from ovary 3 Cells fused Cultured mammary cells 3 4 Grown in Nucleus removed Nucleus from mammary cell culture Early embryo 5 Implanted in uterus of a third sheep Surrogate mother 6 Embryonic development RESULTS Lamb (“Dolly”) genetically identical to mammary cell donor Problems Associated with Animal Cloning • Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs • 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 The aim of stem cell research is to supply cells for the repair of damaged or diseased organs Embryonic stem cells Adult stem cells Early human embryo at blastocyst stage (mammalian equivalent of blastula) From bone marrow in this example Cells generating all embryonic cell types Cells generating some cell types Cultured stem cells Different culture conditions Different types of differentiated cells Liver cells Nerve cells Blood cells Concept 20.4: The practical applications of DNA technology affect our lives in many ways • Many fields benefit from DNA technology and genetic engineering: – Diagnosis of Diseases – Human Gene Therapy – Pharmaceutical Products • Synthesis of Small Molecules for Use as Drugs • Protein Production in Cell Cultures • Protein Production by “Pharm” Animals and Plants Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Human Gene Therapy • Gene therapy is the alteration of an afflicted individual’s genes Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings The practical applications continued… • Many fields benefit from DNA technology and genetic engineering: – Forensic Evidence and Genetic Profiles – Environmental Cleanup – Agricultural Applications (GMO’s) • Animal Husbandry • Genetic Engineering in Plants 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. Forensic Evidence and Genetic Profiles short tandem repeats (STRs), which are variations in the number of repeats of specific DNA sequences (use PCR to amplify) Source of sample STR marker 1 STR marker 2 STR 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. • 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