DNA Technologies PDF

CHAPTER 18 DNA Technologies Peter J. Russell Paul E. Hertz Beverly McMillan www.cengage.com/biology/russell Some Genetically Modified Organisms (GMOs) DNA Technologies Techn...

CHAPTER 18 DNA Technologies Peter J. Russell Paul E. Hertz Beverly McMillan www.cengage.com/biology/russell Some Genetically Modified Organisms (GMOs) DNA Technologies Techniques used to isolate, purify, analyze, and manipulate DNA sequences are known as DNA technologies Scientists use DNA technologies both for basic research into the biology of organisms and for applied research The use of DNA technologies to alter genes for practical purposes is called genetic engineering Biotechnology Genetic engineering is part of biotechnology, which is any technique applied to biological systems or living organisms to make or modify products or processes for a specific purpose Biotechnology also includes non-DNA technologies such as the use of yeast to brew beer and the use of bacteria to make yogurt and cheese 18.1 DNA Cloning DNA cloning is a method for producing many copies of a piece of DNA When DNA cloning involves a gene, it is called gene cloning One common method for cloning a gene of interest is to insert it into plasmids, producing recombinant DNA molecules The plasmids are inserted into bacteria, which replicate the recombinant DNA as they grow and divide Cloning DNA Fragments in a Bacterial Plasmid Gene of Plasmid interest from bacterium Cell 1 2 Inserted 3 genomic DNA fragment Recombinant DNA molecules Figure 18-1a, p. 39 Cloning DNA Fragments in a Bacterial Plasmid Inserted genomic DNA fragment Recombinant DNA molecules 4 5 Bacterium Bacterial chromosome Progeny 6 bacteria Figure 18-1b, p. 39  Gene of Plasmid from bacterium interest Cell 2 1 Inserted 3 genomic DNA fragment Recombina nt DNA molecules 4 Bacteriu m Bacterial chromosome Progeny bacteria 5 Stepped Art Figure 18-1, p. 39 Cloned DNA in Research Cloned DNA may be used in basic research to study gene structure or function, including how its expression is regulated, and the nature of the gene’s product Cloned DNA may be used in applied research for medical, forensic, agricultural, or commercial applications: Gene therapy Diagnosis of genetic diseases Production of pharmaceuticals, Production of genetically modified animals and plants Modification of bacteria to clean up toxic waste Restriction Enzymes Bacterial enzymes called restriction endonucleases (restriction enzymes) are used to join two DNA molecules from different sources Restriction enzymes recognize specific DNA sequences (restriction sites) and cut the DNA at specific locations within those sites The DNA fragments produced by a restriction enzyme are known as restriction fragments Restriction Enzymes (cont.) Restriction enzymes cut DNA at a specific restriction site The sequence of nucleotides (read in the 5′→3′ direction) are the same on both strands (symmetrical) The DNA fragments have single-stranded ends (sticky ends) that hydrogen-bond with complementary sticky ends on other DNA molecules cut with the same enzyme The sugar–phosphate backbones of the DNA strands are sealed by DNA ligase (ligation) Generation of a Recombinant DNA Molecule Restriction site for EcoRI DN A 1 Sticky end Sticky Another DNA fragment end 2 produced by EcoRI digestion Nick in sugar– phosphate backbone EcoRI EcoRI restriction restriction site site 3 Recombinant DNA Figure 18-2, p. 39  Restriction site for EcoRI DNA 1 Sticky end Another DNA Sticky fragment end 2 produced by EcoRI digestion Nick in sugar– phosphate backbone EcoRI EcoRI restriction restriction site site 3 Stepped Art Recombinant DNA molecule Figure 18-2, p. 39 Research Method: Cloning Genes in a Plasmid Vector lacZ+ Gene of Restrict gene interest ion site Plasm Origin id Cel of clonin amp l R replicati g gen on (ori) vecto r e DNA fragments with Plasmid cloning vectors cut with a restriction enzyme to produce sticky ends Inserted DNA Inserted DNAsticky ends Resealed plasmid fragment with fragment without cloning vector gene of interest gene of interest with no inserted DNA fragment Recombinant Nonrecombinant plasmids plasmid Figure 18-3a, p. 39  Recombinant Nonrecombinant plasmids plasmid Selection: Bacteria transformed with Bacteria not Transformed plasmids transformed with bacteria grow on Untransformea plasmid medium with d bacterium ampicillin because can’t grow on of ampR gene on medium plasmid containing Screening: ampicillin Blue colony contains bacteria with a nonrecombinant plasmid; that is, the lacZ+ gene is Plate of intact. growth White colony contains medium bacteria containing For storage, with a recombinant plasmid,ampicillin and bacteria with that is, the vector with an X-gal recombinant inserted DNA fragment. The plasmids are white colonies are screened transferred to to identify the colony with microwell the gene of interest. plates containing growth medium. Figure 18-3, p. 39 Polymerase Chain Reaction (PCR) The polymerase chain reaction (PCR) produces an extremely large number of copies of a specific DNA sequence without having to clone the sequence in a host organism PCR is essentially DNA replication in which a DNA polymerase replicates only a portion of a DNA molecule The primers used in PCR are designed to isolate the sequence of interest – by cycling 20 to 30 times through a series of steps, PCR amplifies the target sequence, producing millions of copies Research Method: PCR Cycle 1 Cycle 2 Cycle 3 Produces 2 molecules Produces Produces 4 molecules 8 molecules Target DNA sequen Templat containi ce e DNA ng primer target DNA New s sequenc primer DNA These 2 e to be molecule amplifie s match d target DNA New DNA primer DNA sequenc e Target Templat sequence e Figure 18-6, p. 39 Gel Electrophoresis Gel electrophoresis is a technique that separates DNA, RNA, or protein molecules in a gel subjected to an electric field –based on size, electrical charge, or other properties PCR results can be compared using agarose gel electrophoresis – the size of the amplified DNA is determined by comparing the position of the DNA band with the positions of bands of a DNA ladder Research Method: Agarose Gel Electrophoresis Well in PCR Micropipettor gel for products adding marker DNA Lane with placing already marker DNA loaded fragments to Agaro well DNA sample to wells fragments se gel Buffer solutio n Gel box Figure 18-7, p. 40 Key Terms Genetic engineering Genomic DNA library DNA cloning cDNA Gene cloning cDNA library Recombinant DNA DNA hybridization Restriction enzyme Polymerase chain Ligation reaction (PCR) DNA ligase Agarose gel electrophoresis Cloning vector 18.2 Applications of DNA Technologies DNA technologies are used in research: Cloning genes to determine their structure, function, and regulation of expression Manipulating genes to determine how their products function in cellular or developmental processes Identifying differences in DNA sequences among individuals in ecological studies DNA technologies also have practical applications: Medical and forensic detection, modification of animals and plants, and manufacture of commercial products Molecular Testing for Human Genetic Diseases Many genetic diseases are caused by defects in enzymes or other proteins that result from mutations at the DNA level Scientists can often use DNA technologies to develop molecular tests for those diseases Normal and Sickle-Cell Mutant Alleles β-Globin gene 175 201 Normal bp bp allele Mst II Mst II Mst II 376 Sickle-cell bp mutant allele Mst II Mst II Region of probe used to screen for sickle-cell mutation Figure 18-8, p. 40 DNA Fingerprinting Each human has unique combinations and variations of DNA sequences known as DNA fingerprints (except identical twins) DNA fingerprinting is a technique used to distinguish between individuals of the same species using DNA samples DNA fingerprinting is commonly used for distinguishing human individuals in forensics and paternity testing Principles of DNA Fingerprinting In DNA fingerprinting, PCR is used to analyze DNA variations at various loci in the genome In the US, 13 loci in noncoding regions of the genome are the standards for PCR analysis Each locus is an example of a short tandem repeat (STR) sequence (or microsatellite) – a short sequence of DNA repeated in series, with each repeat 2-6 bp Principles of DNA Fingerprinting (cont.) Each locus has a different repeated sequence, and the number of repeats varies among individuals in a population As a further source of variation, a given individual is either homozygous or heterozygous for an STR allele Because each individual has an essentially unique combination of alleles, analysis of multiple STR loci can discriminate between DNA of different individuals Using PCR to Obtain a DNA Fingerprint for an STR Locus A. Alleles at an STR locus STR locus Left PCR DNAprimer 9 repeats Right PCR primer 3 different 11 repeats alleles 15 repeats Figure 18-10a, p. 40 Using PCR to Obtain a DNA Fingerprint for an STR Locus B. DNA fingerprint Cells of analysis of the three STR locus by individu PCR als Extract genomic DNA and use PCR to amplify the alleles of the STR locus. Analyze PCR products by gel electrophoresis Positions corresponding to alleles of STR locus Figure 18-10b, p. 40 DNA Fingerprinting in Forensics DNA fingerprints are routinely used to identify criminals or eliminate suspects in legal proceedings DNA fingerprints might be prepared from hair, blood, or semen found at the scene of a crime DNA fingerprinting of stored forensic samples has led to the release of persons wrongly convicted of rape or murder Typically, the evidence is presented in terms of probability that a DNA sample came from a random individual DNA Fingerprinting in Paternity and Ancestry DNA fingerprints are widely used as evidence of paternity because parents and their children share common alleles DNA fingerprints are also used to confirm the identity of human remains DNA fingerprints have been used to investigate pathogenic E. coli in hamburger meat, in cases of wildlife poaching, to detect genetically modified organisms, and to compare the DNA of ancient organisms with present-day descendants Genetic Engineering Genetic engineering uses DNA technologies to modify genes of a cell or organism – organisms that receive genes from an external source (transgenes) are called transgenic Genetic engineering has been used to produce proteins used in medicine and research; to correct hereditary disorders; and to improve animals and crop plants Some people have ethical concerns, or fear that the methods may produce toxic or damaging foods, or release dangerous and uncontrollable organisms Engineering Bacteria to Produce Proteins Engineering E. coli to make a foreign protein: A cDNA copy of a eukaryotic gene for the protein is cloned from the appropriate organism The gene is inserted into an expression vector (plasmid) which contains regulatory sequences that allow transcription and translation of the gene The recombinant plasmid is transformed into E. coli cDNA is expressed in E. coli, transcribed, and translated to make the encoded eukaryotic protein The protein is extracted from bacterial cells and purified, or purified from the culture medium  Using an Expression Vector Start Stop Ribosome Restrictio to Synthesize a Eukaryotic codon codon binding n sites for Protein mRNA AUG UAG site cloning The E. coli for sequence Promot Transcripti machinery eukaryo er on transcribes the tic gene Reverse terminator gene Expression transcriptas and translates the vector for e mRNA to produce ampR E. coli the eukaryotic cDNA gene Origin of protein, copy of replication (ori) which is then mRNA Insertion purified. of cDNA into expression E. Transcrip Promoter vector Terminator coli tion AUG UAG mRN Expression Transform A Translati vector with into E. coli on eukaryotic cDNA inserted Ribosome Eukaryotic binding site protein igure 18-11, p. 405 Genetic Engineering of Animals Several methods are used to introduce a gene of interest into animal cells The gene may be introduced into germ-line cells, which develop into sperm or eggs – the introduced gene is passed from generation to generation The gene may be introduced into differentiated body cells (somatic cells) that do not produce sperm or eggs – the gene is not transmitted from generation to generation Stem Cells Stem cells are cells capable of undergoing many divisions in an undifferentiated state, but also have the ability to differentiate into specialized cell types Embryonic stem cells are found in an early-stage embryo (blastocyst) and can differentiate into all of the tissue types of the embryo Adult stem cells function to replace specialized cells in various tissues and organs Introduction of Genes into Stem Cells In mice, transgenes are introduced into embryonic stem cells, which are then injected into early-stage embryos The stem cells differentiate into a variety of tissues along with embryonic cells, including sperm and egg cells Males and females are bred, leading to offspring that contain one or two copies of the introduced gene A “knockout mouse” is a homozygous recessive that receives two copies of a gene altered to a nonfunctional state Research Method: Gene Introduction by Embryonic Germ-Line Cells Germ-line cells derived from mouse embryo Transgene in expression vector Cell with transgene Figure 18-12a, p. 40 Research Method: Gene Introduction by Embryonic Germ-Line Cells Pure population of transgenic cells Mice have transgenic cells in body regions including germ line Genetically engineered offspring —all cells transgenic Figure 18-12b, p. 40 Genetically Engineered Giant Mouse Figure 18-13, p. 40 Gene Therapy Gene therapy is the introduction of a normal gene into particular cell lines to correct genetic disorders Germ-line gene therapy is the experimental introduction of a gene into germ-line cells of an animal Humans are treated with somatic gene therapy Somatic cells are cultured and transformed with an expression vector containing the transgene Modified cells are reintroduced into the body Gene Therapy in Humans Somatic gene therapy has been successfully used to treat specific cases of adenosine deaminase deficiency (ADA), and sickle-cell disease Other somatic gene therapy trials have ended badly: A teenage patient died as a result of a severe immune response to the viral vector being used Several children in gene therapy trials using retrovirus vectors have developed a leukemia- like condition Turning Domestic Animals into Protein Factories Genetic engineering can turn animals into pharmaceutical factories for production of proteins used to treat human diseases or other medical conditions (e.g. clotting factor) Most animals are engineered to produce proteins in milk, making purification easy, and harmless to the animals Pharming projects are underway for proteins to treat cystic fibrosis, collagen for wrinkles, human milk proteins for infant formulas, and normal hemoglobin for blood transfusions Producing Animal Clones 1997: Ian Wilmut and Keith Campbell successfully cloned a sheep (“Dolly”) using a somatic cell from an adult sheep Several commercial enterprises now provide cloned copies of champion animals Cloned animals often suffer from abnormal conditions – genes may be lost or may be expressed abnormally Molecular studies show that the expression hundreds of genes in the genomes of clones may be regulated abnormally Experimental Research: The First Cloned Mammal Adult white-faced Adult black-faced ewe (donor) ewe Micropipet 1. Diploid cell was te isolated from mammary gland of Nucleus adult white-faced ewe and propagated in 2. Nucleus was tissue culture. removed from 3. Mammary gland cell was fused unfertilized with enucleated egg of black- egg cell. faced ewe. 4. Cells were cultured to produce a cluster that was implanted into uterus of adult black-faced ewe. 5. Embryo developed in surrogate mother. Figure 18-14, p. 40 Genetic Engineering of Plants Plants are engineered for increased resistance to pests and disease; greater tolerance to heat, drought, and salinity; larger crop yields; faster growth; and resistance to herbicides Individual adult cells of some plants can be altered by the introduction of a desired gene, then grown in cultures into a multicellular mass of cloned cells called a callus The callus forms a transgenic plant with the introduced gene in each cell Research Method: Using the Ti Plasmid to Produce Transgenic Plants Agrobacterium tumefaciens T DNA Bacterial chromosom e Restriction site T DNA Ti plasmi d DNA fragment with gene of interest Recombinan t plasmid Figure 18-15a, p. 40 Agrobacterium tumefaciens disarmed so cannot induce tumors Plant cell (not to scale) Nucleu s T DNA with gene of interest integrated into plant cell chromosome Regenerate d transgenic plant Figure 18-15b, p. 40 Plant Genetic Engineering Projects Genetic engineering is used to produce transgenic crops – at least two-thirds of the processed, plant- based foods sold at many national supermarket chains contain transgenic plants Crops such as corn, cotton, and potatoes have been modified for insect resistance by introduction of the bacterial gene for Bt toxin, a natural pesticide Papaya and squash have been genetically engineered for virus resistance Plant Engineering Projects (cont.) Several crops have been engineered for resistance to herbicides – most corn, soybean, and cotton plants grown in the US are glyphosate-resistant (“Roundup-ready”) varieties Crop plants are also being engineered to alter nutritional qualities – “golden rice” contains genes for synthesis of β-carotene, a precursor of vitamin A Plant pharming of transgenic plants to produce medically valuable products is being developed Genetically Engineered Golden Rice Containing β-carotene Genetically engineered Regular rice golden rice containing β-carotene Figure 18-16, p. 41 Public Concerns About Genetic Engineering When recombinant DNA technology was developed, one key concern was that a bacterium carrying a recombinant DNA molecule might escape into the environment, transfer the recombinant molecule to other bacteria, and produce new, potentially harmful, strains To address these concerns, US scientists drew up comprehensive safety guidelines for recombinant DNA research in the United States