Biotechnology Notes PDF

Topic 11: Biotechnology © 2017 Cengage Learning. All Rights Reserved. A Molecular Biology Laboratory © 2017 Cengage Learning. All Rights Reserved. Why It Matters… Techniques used to isolate, purify, analyze, and manipulate DNA sequences...

Topic 11: Biotechnology © 2017 Cengage Learning. All Rights Reserved. A Molecular Biology Laboratory © 2017 Cengage Learning. All Rights Reserved. Why It Matters… 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 © 2017 Cengage Learning. All Rights Reserved. Biotechnology Genetic engineering is part of biotechnology – 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 Biotechnology today often includes genomics (the characterization of whole genomes) and bioinformatics tools (application of mathematics and computer science to biological data) © 2017 Cengage Learning. All Rights Reserved. Key DNA Technologies for Making Genetically Altered Organisms 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 © 2017 Cengage Learning. All Rights Reserved. Genetically Modified Organisms Any organism that has its genome altered to change a genetic trait or traits is a genetically altered organism Genetically modified organisms (GMOs) have their genomes specifically engineered to introduce or change a genetically controlled trait GMOs contain recombinant DNA—DNA fragments from two or more different sources that have been joined together to form a single molecule © 2017 Cengage Learning. All Rights Reserved. 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 or diagnosis of genetic diseases Production of pharmaceuticals Production of genetically modified animals and plants Modification of bacteria to clean up toxic waste © 2017 Cengage Learning. All Rights Reserved. Overview of Gene Cloning Gene of Plasmid interest from bacterium C ell 1 Isolate gene 2 Cut a circular of interest from bacterial plasmid the genome. to make it linear. 3 Insert the gene of interest into the plasmid to make a Inserted recombinant DNA gene of molecule, here a interest recombinant plasmid. from genome 4 Introduce recombinant plasmid into a bacterial cell. As the genetically altered bacterium grows and divides, the plasmid replicates, cloning the gene of interest (or other DNA region of interest) it carries. The cloned gene can be isolated and purified, and used in experiments. Bacterium Bacterial chromoso me Progeny bacteria 5 Cloning genes are used for basic research on genes and proteins to understand their structure, function, and regulation, and for applied research such as modification of animals and plants, and the manufacture of commercial products, including pharmaceuticals. © 2017 Cengage Learning. All Rights Reserved. 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 © 2017 Cengage Learning. All Rights Reserved. Restriction Enzymes (cont'd.) 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) © 2017 Cengage Learning. All Rights Reserved. Generation of a Recombinant Plasmid Bacteri al plasmi d Restriction site for EcoRI 5 3 DNA ' 3 ' ' 5 1 EcoRI restriction ' enzyme cleaves sugar– phosphate backbones of a Sticky bacterial plasmid at end the arrows. 5 3 5 ' ' 5 ' 3 3' 3 ' ' 5' ' Sticky end 2 DNA fragments Another DNA fragment with the same produced by EcoRI sticky ends can pair. Shown here is 5 digestion a DNA fragment ' 3' 5 inserting between 3' ' the two cut ends of the EcoRI-digested bacterial plasmid. Nick in sugar– phosphate backbone 3' 3' 5' 5 5' 3' ' 5' 3 5' 5' ' 3' 3' 3 Nicks in sugar– phosphate backbones are sealed by DNA EcoRI EcoRI ligase. The restrictio restrictio recombinant DNA n site n site molecule has an EcoRI restriction 5 site at each ' junction. 3 Recombinant DNA 3' ' molecule 5' Recombina nt plasmid © 2017 Cengage Learning. All Rights Reserved. Bacterial Plasmids as Cloning Vectors Bacterial plasmids are examples of cloning vectors – DNA molecules into which a DNA fragment is inserted to form a recombinant DNA molecule for the purpose of cloning Plasmid cloning vectors are engineered with two genes used to locate bacteria that incorporate recombinant plasmids: The ampR gene encodes an enzyme that breaks down the antibiotic ampicillin The lacZ+ gene encodes β-galactosidase, which hydrolyzes the sugar lactose © 2017 Cengage Learning. All Rights Reserved. Cloning a Gene of Interest DNA fragments and plasmid, both cut within the lacZ+ gene with the same restriction enzyme, are mixed together with DNA ligase to produce recombinant plasmids DNA molecules are transformed into ampicillin- sensitive, lacZ– E. coli, which are spread on a plate containing ampicillin and the β-galactosidase synthetic substrate X-gal Bacteria that have been transformed with recombinant plasmids are identified by blue-white screening © 2017 Cengage Learning. All Rights Reserved. Research Method: Identifying a Plasmid with a Gene of Interest Identifying a Recombinant Plasmid Containing a Gene of Interest Purpose: To identify a recombinant plasmid containing a gene of interest from a ligation reaction mixture containing a bacterial cloning vector and a DNA fragment containing the Protocol: KEY gene of interest, each digested Inserted DNA fragment with gene of interest with the same restriction enzyme. Resealed plasmid cloning vector (red) with no inserted DNA fragment Restriction site lacZ+ gene 1. The ligation reaction produces Plasmid recombinant plasmids (the only products Cloning with the gene of interest), nonrecombinant vector plasmids, and joined pieces of genomic ampR Origin of DNA (not shown). gene replication (ori) Recombinant plasmid Nonrecombinant plasmid 2. Transform ampicillin-sensitive, lacZ2 E. coli (which cannot make b-galactosidase) with a sample of the ligation reaction. In this step, some bacteria will take up DNA whereas others will not. Bacteria transformed with Bacteria not transformed plasmids with a plasmid or Selection: transformed with gene Bacteria transformed with plasmids fragments grow on medium containing ampicillin because of ampR gene on plasmid. Untransformed Screening: bacteria or bacteria 3. Spread the bacterial cells on a plate of growth medium containing Blue colony contains transformed with ampicillinand X-gal, and incubate the plate until colonies appear. bacteria with a gene fragments nonrecombinant plasmid; cannot grow on that is, the lacZ + medium containing gene is intact. ampicillin. White colony contains bacteria with a recombinant plasmid, that is, Plate of growth the vector with an inserted DNA medium containing fragment, in this case the gene of ampicillin and X-gal interest. Interpreting the Results: All of the colonies on the plate contain plasmids because the bacteria that form the colonies are resistant to theampicillin present in the growth medium. Blue-white screening distinguishes bacterial colonies with nonrecombinant plasmids from those with recombinant plasmids. Bacteria in blue colonies contain nonrecombinant plasmids. These plasmids have intact lacZ1 genes and produce b-galactosidase, which changes X-gal to a blue product. Bacteria in white colonies contain recombinant plasmids. Each recombinant plasmid has a DNA fragment (in this example, the gene of interest) inserted into the lacZ1 gene, so b-galactosidase cannot be produced. As a result, bacteria with recombinant plasmids cannot convert X-gal to the blue product and the colonies are white. Culturing a white colony produces large quantities of the recombinant plasmid that can be isolated and purified for analysis and/or manipulation of the gene. © 2017 Cengage Learning. All Rights Reserved. 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 © 2017 Cengage Learning. All Rights Reserved. Research Method: PCR Cycle 1 Cycle 2 Cycle 3 Produces 2 molecules Produces Produces 4 molecules 8 molecules Target DNA sequence Template containing DNA target primers sequence DNA New to be primer DNA These 2 amplified molecules match target DNA DNA New sequence primer DNA Target Template sequence © 2017 Cengage Learning. All Rights Reserved. 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 © 2017 Cengage Learning. All Rights Reserved. Research Method: Agarose Gel Electrophoresis Separation of DNA Fragments by Agarose Gel Electrophoresis Purpose: Gel electrophoresis separates DNA molecules, RNA molecules, or proteins according to their sizes, electrical charges, or other properties through a gel in an electric field. Different gel types and conditions are used for different molecules and types of applications. A common gel for separating large DNA fragments is made of agarose. PCR products Micropipettor Lane with Protocol: Well in gel already loaded to wells adding marker for placing marker DNA DNA DNA fragments to fragments sample well – – – Courtesy of Janis Shampay Agaros e Gel Buffer solutio n + + + Gel box 1. Prepare a gel consisting of a 2. Load DNA sample solutions, 3. Apply an electric current 4. Stain the gel with a thin such as to the gel; the negatively DNA-binding dye. The slab of agarose and place it in PCR products, into wells of the charged DNA fragments dye fluoresces under a gel box in between two gel, migrate to the positive UV light, enabling the electrodes. The gel has wells alongside a well loaded with a pole. DNA bands to for placing the DNA samples DNA Shorter DNA fragments be seen and to be analyzed. Add buffer to marker ladder (DNA fragments migrate faster than longer photographed. cover the gels. of DNA fragments. At the Shown is an actual gel known sizes). All samples have completion of separation, photograph of the a dye DNA results added to help see the liquid fragments of the same of PCRs on the same when length DNA loading the wells. The dye have formed bands in the sample with four migrates gel. different during electrophoresis, At this point, the bands are pairs of primers, each Interpreting the Results: Agarose gel electrophoresis separates DNA fragments according to their length. enabling the invisible. with a different progress of electrophoresis to predicted size for the The lengths of the DNA fragments being analyzed are determined by measuring their migration distances and be PCR products. followed.) comparing those distances to a calibration curve of the migration distances of the bands of the DNA marker ladder, which have known lengths. For PCR, agarose gel electrophoresis shows whether DNA of the correct length was amplified. For restriction enzyme digests, this technique shows whether fragments are produced as expected. © 2017 Cengage Learning. All Rights Reserved. Review of Key Concepts in This Section Gene cloning Recombinant DNA Restriction enzyme (restriction endonuclease) Ligation DNA ligase Cloning vector Polymerase chain reaction (PCR) Agarose gel electrophoresis © 2017 Cengage Learning. All Rights Reserved. Applications of Genetically Altered Organisms 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 © 2017 Cengage Learning. All Rights Reserved. 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 © 2017 Cengage Learning. All Rights Reserved. Engineering Bacteria to Produce Proteins Engineering E. coli to make a foreign protein: The protein-coding sequence of a 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 The inserted gene 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 © 2017 Cengage Learning. All Rights Reserved.  Using an Expression Vector to Start Stop Ribosome Restriction Synthesize a Eukaryotic Protein codon codon binding site sites for mRNA for AUG UAG sequence cloning The E. coli machinery eukaryotic Promoter Transcription transcribes the gene gene terminator and translates the Reverse mRNA to produce Expression transcriptase the eukaryotic protein, vector for which is then purified. ampR E. coli cDNA copy gene Origin of of mRNA replication Insertion (ori) of cDNA into expression vector E. coli Transcription Promoter Terminator AUG UAG mRNA Expression Transform into Translation vector with E. coli eukaryotic cDNA inserted Ribosome Eukaryotic binding site protein Figure 18-11, p. 405 Cloning Eukaryotic Genes in Bacteria Most eukaryotic protein-coding genes have introns, which are absent in bacterial and most archaeal protein-coding genes After mRNA has been processed in eukaryotes, a double-stranded DNA copy can be made by reverse transcription The enzyme reverse transcriptase can make this copy and is used in reverse transcriptase-PCR This complementary DNA (cDNA) copy can then be cloned and expressed in bacterial cells © 2017 Cengage Learning. All Rights Reserved. Research Method: Reverse Transcription Synthesis of DNA from mRNA Using Reverse Transcriptase Purpose: To produce double-stranded, complementary DNA (cDNA) copies of mRNA molecules isolated from cells. Protocol: mRN A 1. Isolate mRNAs from cells. One mRNA is shown. Poly(A) tail mRNA 3 5' ' 2. Add primer of a short ' UTR ' UTR 5' 3' sequence of T DNA nucleotides UTR UTR (oligo(dT)). Primer base-pairs to poly(A) tail of mRNA 3. Reverse transcriptase uses DNA 5 3 precursors to synthesize a DNA ' 3 ' copy of the mRNA in the 5'-to-3' ' 5 Oligo(dT) ' direction. The result is a hybrid primer nucleic acid molecule consisting of the mRNA base paired with a DNA strand. mRNA 3 5' ' DNA 5 4. An RNase enzyme degrades the 3' ' mRNA strand, leaving a single strand of DNA. DNA 5 3' ' 5. DNA polymerase uses DNA precursors to synthesize the second strand of DNA. Experimentally different methods are available for the use of primers in this reaction. The Doubl 5 3 result is a double-stranded e- stranded' ' complementary DNA (cDNA) copy 3' cDNA 5 of the starting mRNA. ' © 2017 Cengage Learning. All Rights Reserved. Genetic Engineering of Animals Gene targeting is the knocking out, replacement, or addition of a gene in a genome Gene targeting methods have been developed for a number of model animals Many methods require the use of stem cells to produce the GMO © 2017 Cengage Learning. All Rights Reserved. Stem Cells Stem cells are cells capable of undergoing many divisions in an undifferentiated state, and also have the ability to differentiate into specialized cell types Adult stem cells function to replace specialized cells in various tissues and organs These cells are multipotent—they have a restricted ability to produce only certain 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 These cells are pluripotent © 2017 Cengage Learning. All Rights Reserved. Gene Targeting in Mice 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 © 2017 Cengage Learning. All Rights Reserved. Research Method: Making a Knockout Mouse Making a Knockout Mouse Blastocyst from brown-fur mouse Purpose: Make a transgenic mouse in which a specific gene has been knocked out ES cells (deleted) so that the function of that gene is lost. Protocol: Cell culture dish 1.Extract ES cells from blastocyst of an agouti (mottled, grayish brown fur color) mouse neo R gene DNA construct and grow them in cell culture. with neomycin-resistance gene flanked by 2a.Transform the ES cells with a DNA construct consisting of a selectable sequences from the marker, here the neomycin-resistance (neoR) gene (red), flanked by end ends of the mouse gene of interest sequences of the mouse gene of interest. The construct migrates to the Genomic copy of mouse nucleus and the mouse gene sequences align with the homologous gene of interest sequences of a genomic copy of the mouse gene, allowing crossing-over Genomic copy of mouse to occur... an gene—will be degraded d 2b.... The outcome of crossing-over is the replacement of the genomic copy Genomic copy of gene of the gene with the neoR-containing DNA sequence. The chromosomal replaced with construct allele of the mouse gene is now nonfunctional. ES cells with normal gene 3. Select for the ES cells in which a normal gene has been replaced by the replaced with neo R gene growing in media neoRcontaining containing neomycin DNA sequence by growing cells in media containing neomycin. 4. Inject genetically engineered ES cells into blastocysts from white mice. Blastocyst from white-fur mouse 5. Implant the blastocysts into white, surrogate (foster) mother. White-fur, 6.Some progeny mice will be white; others will be chimeric mice with patches of agouti surrogate mother fur. (Agouti fur is genetically dominant to white.) Chimeric mice have many cells derived from the original white mouse blastocyst, but some cells derived from the agouti mouse-derived genetically engineered ES cells that were introduced into the Chimeric mouse blastocyst in step 4. 7. Mate chimeric mice with white mice. If the chimeric mouse parent as gonads derived Transgenic mouse from the genetically engineered stem cells, all offspring will have agouti fur. The heterozygous for gene knockout agouti offspring are heterozygous for the gene knockout. 8. Interbreed the heterozygous knockout mice. Perform DNA testing on agouti progeny to identify those homozygous for the gene knockout. Transgenic mouse homozygous for gene knockout Outcome: The result is a mouse in which both chromosomal alleles of a specific gene of interest have been knocked out. The effects of the loss of function of that gene can then be studied. © 2017 Cengage Learning. All Rights Reserved. Focus on Research: Programmable RNA- Guided Genome Editing System CRISPR loci and cas genes together encode an immune system against foreign bacteriophages and plasmids in bacterial and archaeal cells The natural CRISPR-Cas system has been modified to be a programmable RNA-guided genome editing system for research purposes This technology has been embraced rapidly by research groups for both basic and applied research Example: making gene knockouts is simpler and more time-efficient with CRISPR-Cas than traditional methods © 2017 Cengage Learning. All Rights Reserved. Programmable RNA-Guided Genome Editing Target DNA (invading Cas foreign DNA) 5 3 ' Cut ' 3 5 ' ' sgRNA © 2017 Cengage Learning. All Rights Reserved. Gene Therapy Gene therapy is the introduction of a normal gene into particular cell lines to correct genetic disorders Germline gene therapy is the experimental introduction of a gene into germline cells of an animal This type of therapy is not allowed in humans 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 © 2017 Cengage Learning. All Rights Reserved. Gene Therapy in Humans Somatic gene therapy has been successfully used to treat specific cases of adenosine deaminase deficiency (ADA) 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 © 2017 Cengage Learning. All Rights Reserved. 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 © 2017 Cengage Learning. All Rights Reserved. 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 © 2017 Cengage Learning. All Rights Reserved. Experimental Research: The First Mammal Clone Adult white-faced ewe Adult black-faced ewe (donor) Micropipette Nucleus 1. Diploid cell was isolated from mammary 2. Nucleus was removed from gland of adult unfertilized egg of black-faced ewe. white-faced ewe and propagated in tissue culture. 3. Mammary gland cell was fused with nucleated egg cell. 4. Cells were cultured to produce a cluster that was implanted into uterus of adult black-faced ewe. 5. Embryo developed in surrogate mother. © 2017 Cengage Learning. All Rights Reserved. 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 © 2017 Cengage Learning. All Rights Reserved. Methods Used to Insert Genes into Plants: The Ti Plasmid A tumor of deciduous trees (crown gall disease) is caused by the bacterium Agrobacterium tumefaciens, which contains a large, circular plasmid – the Ti (tumor-inducing) plasmid Genes on a segment of the Ti plasmid (T DNA) integrate into the plant genome and are expressed; the products stimulate cell growth and division, producing a tumor The Ti plasmid is used as a vector for making transgenic plants (similar to bacterial plasmids used in bacteria) © 2017 Cengage Learning. All Rights Reserved. Research Method: The Ti Plasmid Using the Ti Plasmid of Agrobacterium tumefaciens to Produce Transgenic Plants Purpose: To make transgenic plants. This technique is one way to introduce a transgene into a plant for genetic engineering purposes. Protocol: Agrobacterium T DNA 1. Isolate the Ti plasmid from tumefaciens Agrobacterium tumefaciens. The plasmid contains a segment called T DNA (T = transforming), Bacterial which induces tumors in plants. chromosome Restriction 2. Digest the Ti plasmid with a site T DNA restriction enzyme that cuts within the T DNA. Mix with a gene of interest on a DNA fragment that was produced by Ti digesting with the same enzyme. Use plasmid DNA ligase to join the two DNA molecules together to produce a recombinant plasmid. DNA fragment with gene of 3. Transform the recombinant Ti plasmid interest into a disarmed A. tumefaciens that cannot induce tumors, and use the transformed bacterium to infect cells Recombinan in plant fragments in a test tube. In t plasmid infected cells, the T DNA with the inserted gene of interest excises from the Ti plasmid and integrates into the plant cell genome. Agrobacterium tumefaciens disarmed so cannot induce tumors Plant cell (not to scale) Nucleus T DNA with gene of interest integrated into plant cell chromosome 4. Culture the transgenic plant fragments Regenerate to regenerate whole plants. d Outcome: The plant has been genetically transgenic engineered plant to contain a new gene. The transgenic plant will express a new trait based on that gene, perhaps resistance to an herbicide or production of an © 2017 Cengage Learning. All Rights Reserved. insect 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 © 2017 Cengage Learning. All Rights Reserved. Plant Engineering Projects (cont'd.) 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 © 2017 Cengage Learning. All Rights Reserved. Genetically Engineered Golden Rice Containing β-carotene Regular Genetically engineered rice golden rice containing - carotene Shutterstock.com/CHAIWATPHOTOS Shutterstock.com/Loskutnikov © 2017 Cengage Learning. All Rights Reserved. Molecular Insights: Nutritional Quality of Genetically Modified Food Research Question: Are the metabolomes of genetically modified foods significantly different from their unmodified (transgenic) versions? Conclusion: Genetically modifying tomatoes to delay fruit ripening does not significantly affect the metabolic fingerprint of the tomato fruit other than the fruit-ripening metabolites © 2017 Cengage Learning. All Rights Reserved. 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, U.S. scientists drew up comprehensive safety guidelines for recombinant DNA research in the United States © 2017 Cengage Learning. All Rights Reserved. Public Concerns About Genetic Engineering (cont'd.) Issues include the safety of consuming GMO- containing foods, and possible adverse effects on the environment: GMOs interbreeding with natural species Beneficial insect species such as monarch butterflies feeding on plants with Bt toxins © 2017 Cengage Learning. All Rights Reserved. Global Reactions Different countries have reacted to GMOs in different ways: In the U.S., GMOs are evaluated for potential risk by appropriate government regulatory agencies In the EU, all use of GMOs in the field or in food requires authorization following a careful review process On a global level, the Cartagena Protocol on Biosafety “promotes biosafety by establishing practical rules and procedures for the safe transfer, handling and use of GMOs” © 2017 Cengage Learning. All Rights Reserved. Other Applications of DNA Technologies Some applications for DNA technologies do not involve making or using genetically altered organisms 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 Example: The sickle-cell mutation changes a restriction site in the DNA – cutting the β-globin gene with MstII produces two DNA fragments from the normal gene and one fragment from the mutated gene © 2017 Cengage Learning. All Rights Reserved. Normal and Sickle-Cell Mutant Alleles β-Globin gene Left PCR primer 175 bp 201 bp Normal allele Right PCR MstII MstII MstII primer 376 bp Sickle-cell mutant allele MstII MstII © 2017 Cengage Learning. All Rights Reserved. RFLPs Restriction enzyme-generated DNA fragments of different lengths from the same region of the genome are known as restriction fragment length polymorphisms (RFLPs) RFLPs typically are analyzed using agarose gel electrophoresis The single base-pair mutation in the b-globin gene in sickle-cell anemia is an example of a single- nucleotide polymorphism (SNP) An SNP locus typically has two alleles; by definition to be an SNP, the frequency of the rarer gene must be at least 1% © 2017 Cengage Learning. All Rights Reserved. RFLP Analysis of Sickle-Cell Mutant Alleles Cells from A. PCR-amplified, individual with MstII-digested DNA A B C sickle-cell anemia from sickle-cell anemia individual – Cells from B. PCR-amplified, 376 bp normal MstII-digested individual DNA from normal Analyze digested PCR products individual 201 bp by gel electrophoresis. 175 bp C. PCR- Cell from sickle- amplified, cell trait + MstII-digested individual DNA from (heterozygote) sickle-cell trait PCR-amplified, MstII-digested DNA from the different genotypes individual show different banding patterns after gel electrophoresis: (A) sickle-cell individual—single band of 376 bp; (B) normal Isolate genomic DNA, amplify the β-globin gene region individual—two bands, of 201 bp and 175 bp; (C) sickle-cell by PCR, and digest the resulting DNA fragments with MstII. trait heterozygote—three bands, of 376 bp (from the sickle-cell mutant allele), and 201 bp and 175 bp (both from the normal allele). © 2017 Cengage Learning. All Rights Reserved. DNA Fingerprinting Each human has unique combinations and variations of DNA sequences known as DNA fingerprints (except identical twins) DNA fingerprinting (also called DNA profiling) 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 © 2017 Cengage Learning. All Rights Reserved. Principles of DNA Fingerprinting In DNA fingerprinting, PCR is used to analyze DNA variations at various loci in the genome In the U.S., 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 © 2017 Cengage Learning. All Rights Reserved. Principles of DNA Fingerprinting (cont'd.) 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 © 2017 Cengage Learning. All Rights Reserved. Using PCR to Obtain a DNA Fingerprint A. Alleles at an STR B. DNA fingerprint analysis of the STR locus by locus PCR STR locus A B Left PCR C DNA primer Cells of 9 Right PCR three repeats primer individual s 3 11 different repeats alleles Extract genomic DNA 15 and use PCR repeats to amplify the alleles of the STR locus. Analyze PCR products by gel electrophoresis. A B C Positions corresponding to alleles of STR locus 15 11 9 11, 15, 11, 11 9 9 © 2017 Cengage Learning. All Rights Reserved. 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 © 2017 Cengage Learning. All Rights Reserved. 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 © 2017 Cengage Learning. All Rights Reserved.