© 2015 pearson education, inc. introduction hawaii’s papaya industry seemed doomed just a few...

2015 Pearson Education, Inc. Introduction Hawaiis papaya industry seemed doomed just a few decades ago. A deadly pathogen called the papaya ringspot virus (PRV) had spread throughout the islands. It appeared poised to completely decimate the papaya plant population. Scientists from the University of Hawaii were able to rescue the industry by creating new, genetically engineered PRV-resistant strains of papaya. Today, the papaya industry is once again vibrant, and the vast majority of Hawaiis papayas are genetically modified organisms (GMOs).

GMO Genetically Modified Organisms (GMO): When a gene from one organism is purposely moved to improve or change another organism in a laboratory, the result is a genetically modified organism (GMO). It is also sometimes called "transgenic" for transfer of genes. 84% of corn grown today is genetically modified. Most is used in processed foods and some are used for animal feed. Some strains are drought resistant, others are pest resistant, some are herbicide resistant. 94% of the soy crop in the US is genetically modified. Very few fresh fruits and vegetables in your local grocery store are genetically modified. Potatoes are one of the genetically engineered vegetables available in the United States. Other genetically modified vegetables that have been approved for sale in the U.S. are tomatoes, radicchio, zucchini and yellow squash.

Isnt everything genetically modified????

2015 Pearson Education, Inc. Genes can be cloned in recombinant plasmids Biotechnology- For thousands of years, humans have used microbes to make wine and cheese and selectively bred stock, dogs, and other animals. DNA technology Genetic engineering-

2015 Pearson Education, Inc. Genes can be cloned in recombinant plasmids Gene cloning- Recombinant DNA One source contains the gene that will be cloned. Another source is a gene carrier, called a ______. _________ are small, circular DNA molecules that replicate separately from the much larger bacterial chromosome; they are often used as vectors.

2015 Pearson Education, Inc. Genes can be cloned in recombinant plasmids The steps in cloning a gene: 1._____________is isolated. 2.DNA containing the gene of interest is isolated. 3.Plasmid DNA is treated with a _____________ that cuts in one place, opening the circle. 4.DNA with the target gene is treated with the same enzyme, and many fragments are produced. 5.Plasmid and target DNA __________________.

2015 Pearson Education, Inc. 654321 Plasmid Bacterial chromosome E. coli bacterium A plasmid is isolated. The plasmid is cut with an enzyme The cells DNA is isolated. DNA A cell with DNA containing the gene of interest Gene of interest (gene V) The cells DNA is cut with the same enzyme. Gene of interest The targeted fragment and plasmid DNA are combined. DNA ligase is added, which joins the two DNA molecules. Gene of interest Recombinant DNA plasmid

2015 Pearson Education, Inc. 7 8 9 Genes may be inserted into other organisms. Harvested proteins may be used directly. The recombinant plasmid is taken up by a bacterium through trans- formation. The bacterium reproduces. Recombinant DNA plasmid Recombinant bacterium Clone of cells Gene of interest

2015 Pearson Education, Inc. VISUALIZING THE CONCEPT: Enzymes are used to cut and paste DNA Restriction enzymes recognize a particular short DNA sequence, called a restriction site, and cut both strands of the DNA at precise points in the sequence, yielding pieces of DNA called restriction fragments. Once cut, fragments of DNA can be pasted together by the enzyme DNA ligase.

2015 Pearson Education, Inc. Animation: Restriction Enzymes

2015 Pearson Education, Inc. Restriction enzyme Restriction site DNA Sticky end Gene of interest Sticky end DNA ligase Recombinant DNA Every restriction enzyme recognizes one specific nucleotide sequence (its restriction site). A restriction enzyme always cuts DNA sequences at its restriction site in an identical manner. A piece of DNA from another source (the gene of interest) is cut by the same restriction enzyme. The DNA fragments from the two sources stick together by hydrogen bonding of base pairs. The enzyme DNA ligase creates new covalent bonds that join the backbones of the DNA strands. The result is a piece of recombinant DNA. G AATTC G CTTAA AATTC CTTAA G G G GG G C C C CAATT TTAA GAATTC CTTAAG

2015 Pearson Education, Inc. Recombinant cells and organisms can mass-produce gene products Recombinant cells and organisms constructed by DNA technologies are used to manufacture many useful products, chiefly proteins. Bacteria are often the best organisms for manufacturing a protein product because bacteria have plasmids and phages available for use as gene- cloning vectors, can be grown rapidly and cheaply, can be engineered to produce large amounts of a particular protein, and often secrete the proteins directly into their growth medium.

2015 Pearson Education, Inc. Recombinant cells and organisms can mass-produce gene products Yeast cells are eukaryotes, are easy to grow, have long been used to make bread and beer, can take up foreign DNA and integrate it into their genomes, and are often better than bacteria at synthesizing and secreting eukaryotic proteins.

2015 Pearson Education, Inc. Recombinant cells and organisms can mass-produce gene products Mammalian cells must be used to produce glycoproteins, proteins with chains of sugars attached. Examples include human erythropoietin (EPO), which stimulates the production of red blood cells, factor VIII to treat hemophilia, and tissue plasminogen activator (TPA), used to treat heart attacks and strokes.

2015 Pearson Education, Inc.

Recombinant cells and organisms can mass-produce gene products Pharmaceutical researchers are currently exploring the mass production of gene products by whole animals or plants. Recombinant animals are difficult and costly to produce and may be cloned to produce more animals with the same traits.

2015 Pearson Education, Inc. CONNECTION: DNA technology has changed the pharmaceutical industry and medicine DNA technology, including gene cloning, is widely used to produce medicines and to diagnose diseases. Therapeutic hormones produced by DNA technology include insulin to treat diabetes, human growth hormone to treat dwarfism, and tissue plasminogen activator (TPA), a protein that helps dissolve blood clots and reduces the risk of subsequent heart attacks.

2015 Pearson Education, Inc. CONNECTION: DNA technology has changed the pharmaceutical industry and medicine DNA technology is used to test for inherited diseases, detect infectious agents such as HIV, and produce vaccines, harmless variants (mutants) or derivatives of a pathogen that stimulate the immune system to mount a lasting defense against that pathogen, thereby preventing disease.

2015 Pearson Education, Inc. CONNECTION: Genetically modified organisms are transforming agriculture Since ancient times, people have selectively bred agricultural crops to make them more useful. DNA technology is quickly replacing traditional breeding programs to improve the productivity of agriculturally important plants and animals. Genetically modified organisms (GMOs) contain one or more genes introduced by artificial means. Transgenic organisms contain at least one gene from another species.

2015 Pearson Education, Inc. CONNECTION: Genetically modified organisms are transforming agriculture The most common vector used to introduce new genes into plant cells is a plasmid from the soil bacterium Agrobacterium tumefaciens called the Ti plasmid.

2015 Pearson Education, Inc. Plant cell 213 Agrobacterium tumefaciens DNA containing the gene for a desired trait Ti plasmid Restriction site The gene is inserted into the plasmid. Recombinant Ti plasmid The recombinant plasmid is introduced into a plant cell. DNA carrying the new gene The plant cell grows into a plant. A plant with the new trait

2015 Pearson Education, Inc. CONNECTION: Genetically modified organisms are transforming agriculture GMO crops may be able to help a great many hungry people by improving food production, shelf life, pest resistance, and the nutritional value of crops. Golden Rice, a transgenic variety created in 2000 with a few daffodil genes, produces yellow grains containing beta-carotene, which our body uses to make vitamin A.

2015 Pearson Education, Inc. CONNECTION: Genetically modified organisms are transforming agriculture Genetic engineers are now creating plants that make human proteins for medical use. Pharmaceutical trials currently under way involve using modified rice to treat infant diarrhea, corn to treat cystic fibrosis, safflower to treat diabetes, and duckweed to treat hepatitis. Although promising, no plant-made drugs intended for use by humans have been approved or sold.

2015 Pearson Education, Inc. CONNECTION: Gene therapy may someday help treat a variety of diseases Gene therapy is the alteration of a diseased individuals genes for therapeutic purposes. One possible procedure is the following: 1.A gene from a healthy person is cloned, converted to an RNA version, and then inserted into the RNA genome of a harmless virus. 2.Bone marrow cells are taken from the patient and infected with the recombinant virus. 3.The virus inserts a DNA version of its genome, including the normal human gene, into the cells DNA. 4.The engineered cells are then injected back into the patient.

2015 Pearson Education, Inc. Healthy person 1234 Cloned gene (normal allele) An RNA version of a healthy human gene is inserted into a retrovirus. RNA genome of virus Retrovirus Bone marrow cells are infected with the virus. Viral DNA carrying the human gene inserts into the cells chromosome. Bone marrow cell from the patient Bone marrow The engineered cells are injected into the patient.

2015 Pearson Education, Inc. The analysis of genetic markers can produce a DNA profile DNA profiling is the analysis of DNA samples to determine whether they came from the same individual. In a typical investigation involving a DNA profile: 1.DNA samples are isolated from the crime scene, suspects, victims, or other evidence, 2.selected markers from each DNA sample are amplified (copied many times), producing a large sample of DNA fragments, and 3.the amplified DNA markers are compared, providing data about which samples are from the same individual.

2015 Pearson Education, Inc. Crime scene 231 Suspect 1 Suspect 2 DNA is isolated. The DNA of selected markers is amplified. The amplified DNA is compared.

2015 Pearson Education, Inc. The PCR method is used to amplify DNA sequences Polymerase chain reaction (PCR) is a technique by which a specific segment of a DNA molecule can be targeted and quickly amplified in the laboratory.

2015 Pearson Education, Inc. The PCR method is used to amplify DNA sequences PCR relies upon a pair of short primers, which are chemically synthesized, single-stranded DNA molecules with sequences that are complementary to sequences at each end of the target sequence. One primer is complementary to one strand at one end of the target sequence. The second primer is complementary to the other strand at the other end of the sequence. The primers thus bind to sequences that flank the target sequence, marking the start and end points for the segment of DNA being amplified.

2015 Pearson Education, Inc. The PCR method is used to amplify DNA sequences The basic steps of PCR are as follows: 1.The reaction mixture is heated to separate the strands of the DNA double helices. 2.The strands are cooled. As they cool, primer molecules hydrogen-bond to their target sequences on the DNA. 3.A heat-stable DNA polymerase builds new DNA strands by extending the primers in the 53 direction. These three steps are repeated over and over, doubling the amount of DNA after each three-step cycle.

2015 Pearson Education, Inc. 3355 3355 3355 33 55 55 3355 3355 55 3355 3355 3355 3355 Cycle 1 yields two molecules Cycle 2 yields four molecules Cycle 3 yields eight molecules Additional Cycles Sample DNA Target sequence PrimerNew DNA Heat separates DNA strands. Primers bond with ends of target sequences. DNA polymerase adds nucleotides. 12 3

2015 Pearson Education, Inc. The PCR method is used to amplify DNA sequences Devised in 1985, PCR has had a major impact on biological research and biotechnology. PCR has been used to amplify DNA from fragments of ancient DNA from a mummified human, a 40,000-year-old frozen woolly mammoth, a 30-million-year-old plant fossil, and DNA from fingerprints or from tiny amounts of blood, tissue, or semen found at crime scenes.

2015 Pearson Education, Inc. Gel electrophoresis sorts DNA molecules by size Many DNA technology applications rely on gel electrophoresis, a method that separates macromolecules, usually proteins or nucleic acids, on the basis of size, electrical charge, or other physical properties.

2015 Pearson Education, Inc. Gel electrophoresis sorts DNA molecules by size Gel electrophoresis can be used to separate DNA molecules based on size as follows: 1.A DNA sample is placed at one end of a porous gel. 2.Current is applied, and DNA molecules move from the negative electrode toward the positive electrode. 3.Shorter DNA fragments move through the gel matrix more quickly and travel farther through the gel. 4.DNA fragments appear as bands, visualized through staining or detecting radioactivity or fluorescence. 5.Each band is a collection of DNA molecules of the same length.

2015 Pearson Education, Inc. Gel Power source A mixture of DNA fragments of different sizes Completed gel Longer (slower) molecules Shorter (faster) molecules

2015 Pearson Education, Inc. Short tandem repeat analysis is commonly used for DNA profiling Repetitive DNA consists of nucleotide sequences that are present in multiple copies in the genome. Short tandem repeats (STRs) are short nucleotide sequences that are repeated in tandem, composed of different numbers of repeating units in individuals, that are used in DNA profiling. STR analysis compares the lengths of STR sequences at specific sites in the genome and typically analyzes 13 sites scattered in the genome.

2015 Pearson Education, Inc. STR site 1 STR site 2 Crime scene DNA AGATGATA The number of short tandem repeats match The number of short tandem repeats do not match GATA AGAT Suspects DNA

2015 Pearson Education, Inc. Amplified crime scene DNA Amplified suspects DNA Longer STR fragments Shorter STR fragments

2015 Pearson Education, Inc. CONNECTION: DNA profiling has provided evidence in many forensic investigations DNA profiling is used to determine guilt or innocence in a crime, settle questions of paternity, and probe the origin of nonhuman materials.

2015 Pearson Education, Inc. RFLPs can be used to detect differences in DNA sequences Geneticists have cataloged many single-base-pair variations in the genome. Such a variation found in at least 1% of the population is called a single nucleotide polymorphism (SNP, pronounced snip). SNPs occur on average about once in 100 to 300 base pairs in the human genome, in the coding sequences of genes and in noncoding sequences between genes.

2015 Pearson Education, Inc. RFLPs can be used to detect differences in DNA sequences SNPs may alter a restriction sitethe sequence recognized by a restriction enzyme. Such alterations change the lengths of the restriction fragments formed by that enzyme when it cuts the DNA. A sequence variation of this type is called a restriction fragment length polymorphism (RFLP, pronounced rif-lip). Thus, RFLPs can serve as genetic markers for particular loci in the genome.

2015 Pearson Education, Inc. Cut Restriction enzymes added Sample 1 DNA sample 1 Longer fragments Shorter fragments w Sample 2 DNA sample 2 x y y z yy z x w CCGGCCGG CCGGCCGG CCGGCCGG GC GC GC TGCCTGCC ACGGACGG

2015 Pearson Education, Inc. You should now be able to 1.Explain how plasmids are used in gene cloning. 2.Explain how restriction enzymes are used to cut and paste DNA into plasmids. 3.Explain how DNA technology has helped to produce insulin, growth hormone, and vaccines. 4.Explain how genetically modified organisms (GMOs) are transforming agriculture. 5.Describe the benefits and risks of gene therapy in humans.

2015 Pearson Education, Inc. You should now be able to 6.Describe the benefits and risks of gene therapy in humans. 7.Describe the basic steps of DNA profiling. 8.Explain how PCR is used to amplify DNA sequences. 9.Explain how gel electrophoresis is used to sort DNA and proteins. 10.Explain how short tandem repeats are used in DNA profiling. 11.Explain how restriction fragment analysis is used to detect differences in DNA sequences.

2015 Pearson Education, Inc. Figure 12.UN02 A mixture of DNA fragments A band is a collection of DNA fragments of one particular length Longer fragments move slower Shorter fragments move faster Power source DNA is attracted to + pole due to PO 4 groups

2015 Pearson Education, Inc. Figure 12.UN03 DNA amplified via Bacterial plasmids DNA sample treated with DNA fragments sorted by size via Recombinant plasmids are inserted into bacteria Add Particular DNA sequence highlighted are copied via treated with (a) (b) (c) (d) (e)

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