Chapter 20 Biotechnology Power Point Lecture Presentations for

Chapter 20 Biotechnology Power. Point® Lecture Presentations for Biology Eighth Edition Neil 1 Campbell and Jane Reece Lectures by Chris Romero, updated by Erin Barley with contributions from Joan Sharp Copyright © 2008 Pearson Education, Inc. , publishing as Pearson Benjamin Cummings

Overview: The DNA Toolbox Sequencing of the human genome was completed by 2007 DNA sequencing has depended on advances in technology, starting with making recombinant DNA In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule 2

3 Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes

Fig. 20 -1

Concept 20. 1: DNA cloning yields multiple copies of a gene or other DNA segment To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called DNA cloning Why? To create multiple copies of a gene To produce a protein end product 5

DNA Cloning and Its Applications: A Preview Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome 6

Gene cloning involves using bacteria to make multiple copies of a gene Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA This results in the production of multiple 7 copies of a single gene

Fig. 20 -2 a Bacterium 1 Gene inserted into Cell containing gene of interest plasmid Bacterial chromosome Plasmid Recombinant DNA (plasmid) Gene of interest 2 2 Plasmid put into bacterial cell Recombinant bacterium DNA of chromosome

Fig. 20 -2 b Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed by gene of interest Gene of Interest Copies of gene Protein harvested 4 Basic research and Basic research on gene Gene for pest resistance inserted into plants various applications 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

Using Restriction Enzymes to Make Recombinant DNA Bacterial restriction enzymes cut DNA molecules at specific DNA sequences They protect the bacterial cell by cutting foreign DNA from other organisms or phages Hundreds have been identified, they are highly specific and recognize a particular sort DNA sequence or Restriction site Most restriction sites are symetrical (meaning the same sequence whether it is read 5’-3’ or 3’ to 5’ Usually the bacterial DNA is protected from the enzymes by the addition of methyl groups to adenines or cytosines within the sites normally recognized by the enzyme 10 Animation: Restriction Enzymes

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 These are temporary hydrogen bonds, but can be made permanent by DNA ligase 11

DNA ligase is an enzyme that seals the bonds between restriction fragments(catalyzes covalent bonds that close the sugar-phosphate backbones- like in Okazaki fragments) https: //www. youtube. com/watch? v=8 r. Xizm. Ljeg. I Mechanisms of recombinant DNA 12

Fig. 20 -3 -1 Restriction site DNA 1 5 3 3 5 Restriction enzyme cuts sugar-phosphate backbones. Sticky end symetrical

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

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

Cloning a Eukaryotic Gene in a Bacterial Plasmid 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 Why? Easily isolated from bacteria, Easily manipulated to form recombinant plasmids by insertion of foreign DNA in vitro Multiply quickly https: //www. youtube. com/watch? v=GNMJBMt KKWU plasmids 16

Producing Clones of Cells Carrying Recombinant Plasmids Several steps are required to clone the hummingbird β-globin gene in a bacterial plasmid: WHY would I even care about hummingbird beta-globin? high metabolism, is their hemoglobin different than other organisms with less active metabolisms? can we manipulate metabolisms this way? 17 Animation: Cloning a Gene

How it’s done: The hummingbird genomic DNA and a bacterial plasmid are isolated The plasmid has been engineered to have ampicillin resistance, and a lac. Z which encodes an enzyme to break down lactose Also causes a blue product Only one copy of the restriction site (so will only be cut once) 18

Both are digested with the same restriction enzyme The fragments are mixed DNA ligase is added to bond the fragment sticky ends Some recombinant plasmids now contain hummingbird DNA 19

This is like what you guys did with the p. GLO The DNA mixture is added to bacteria that have been genetically engineered to accept it The bacteria are plated on a type of agar that selects for the bacteria with recombinant plasmids This results in the cloning of many hummingbird DNA fragments, including the β-globin gene 20

Fig. 20 -4 -1 Hummingbird cell TECHNIQUE Bacterial cell amp. R gene lac. Z gene Bacterial plasmid Restriction site Sticky ends Gene of interest Hummingbird DNA fragments

Fig. 20 -4 -2 Hummingbird cell TECHNIQUE Bacterial cell amp. R gene lac. Z gene Bacterial plasmid Restriction site Sticky ends Gene of interest Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids

Fig. 20 -4 -3 Hummingbird cell TECHNIQUE Bacterial cell amp. R gene lac. Z gene Bacterial plasmid Restriction site Sticky ends Gene of interest Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carrying plasmids

Fig. 20 -4 -4 Hummingbird cell TECHNIQUE Bacterial cell amp. R gene lac. Z gene Bacterial plasmid Restriction site Sticky ends Gene of interest Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carrying plasmids RESULTS Colony carrying nonrecombinant plasmid with intact lac. Z gene Colony carrying recombinant plasmid with disrupted lac. Z 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 There a lot of different recombinants made by the previous procedure Each contains specific information Scientists can create their own, or purchase from another source A genomic library that is made using bacteriophages is stored as a collection of phage clones Can have a larger size of DNA inserted into it 25

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

Fig. 20 -5 a Foreign genome cut up with restriction enzyme or Recombinant phage DNA Bacterial clones (a) Plasmid library Recombinant plasmids (b) Phage library 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 28

Fig. 20 -5 b Large plasmid Large insert with many genes BAC clone (c) A library of bacterial artificial chromosome (BAC) clones

A complementary DNA (c. DNA) library is made by cloning DNA made in vitro by reverse transcription of all the m. RNA produced by a particular cell A c. DNA library represents only part of the genome—only the subset of genes transcribed into m. RNA in the original cells 30

Fig. 20 -6 -1 DNA in nucleus m. RNAs in cytoplasm Reverse transcriptase is added to a test Tube containing m. RNA isolated from a cell (to get the m. RNA from a cell-literally a recipe of adding this and that, Homogenizing the cell, centrifuging, taking The RNA pellet out from the bottom and Suspending the RNA in a solution) The reverse transcriptase catalyzes a complementary Strand of the m. RNA to be made, without the introns

Fig. 20 -6 -2 DNA in nucleus m. RNAs in cytoplasm m. RNA Reverse transcriptase Poly-A tail DNA Primer strand

Fig. 20 -6 -3 DNA in nucleus m. RNAs in cytoplasm m. RNA Reverse transcriptase Poly-A tail Degraded m. RNA DNA Primer strand The original m. RNA Is degraded by another Enzyme, so you can isolate different genes of interest, or order The c. DNA of what you are interested in

Fig. 20 -6 -4 DNA in nucleus m. RNAs in cytoplasm m. RNA Reverse transcriptase Poly-A tail Degraded m. RNA DNA polymerase DNA Primer strand

Fig. 20 -6 -5 DNA in nucleus m. RNAs in cytoplasm m. RNA Reverse transcriptase Poly-A tail DNA Primer strand Degraded m. RNA DNA polymerase c. DNA

Screening 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 gene (usually radioactive so it “glows” when you take a photo of it) This process is called nucleic acid hybridization 36

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 37

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 38

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

You can measure the amount of a particular sequence by the amount of radiation that is given off, the higher the radiation, the more of that particular sequence that you labeled you have. In theory: the more message you have, the more protein that results Southern Blot is used to quantify DNA Northern Blot is used to quantify RNA 40

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Expressing Cloned Eukaryotic Genes 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 42

Bacterial Expression Systems Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells Eukaryotic genome extremely large, so using a c. DNA would be better because only has the exons 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 Allows the bacterial host cell to recognize the promoter and then allow expression of the eukaryotic gene 43

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 Yeast (eukaryotic) can handle the larger size Are easy to grow Have plasmids 44

YACs behave normally in mitosis and can carry more DNA than a plasmid Eukaryotic hosts can provide the posttranslational modifications that many proteins require 45

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 46

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 (denature), cooling (annealing), and replication (DNA polymerase)—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules 47

Fig. 20 -8 a 5 TECHNIQUE 3 Target sequence Genomic DNA 3 5

Fig. 20 -8 b 1 Denaturation 5 3 Heating separates The strands 3 5 2 Annealing Cycle 1 yields 2 molecules Primers Cooling allows Primers to bond 3 Extension New nucleotides Special DNA polymerase From thermophiles Add nucleotides

Fig. 20 -8 c Cycle 2 yields 4 molecules

Fig. 20 -8 d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

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 52

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 53 Video: Biotechnology Lab

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Fig. 20 -9 a TECHNIQUE Mixture of DNA molecules of different sizes Power source Anode + – Cathode Gel 1 Each sample put in a different “well”; the gel is floating in a buffer solution; power Is attached and the gel is run for a certain amount of time; the slower the better so the band of DNA separate according to molecular weight Power source The larger molecules Move more slowly; the Shorter ones move Faster; 2 – + Longer molecules Shorter molecules

After the gel is turned off, the gel Is dyed (could be just a dye that Shows under UV light OR the gel Is labeled with radioactive material That shows up. If using the same restriction enzyme, Then this can show the source of the DNA, the lines would be the same if They were from the sample, if The lines are different the DNA came From different genetic sources

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 58

Fig. 20 -10 Normal -globin allele 175 bp Dde. I Sickle-cell allele Large fragment 201 bp Dde. I Normal allele Dde. I Large fragment Sickle-cell mutant -globin allele 376 bp Dde. I 201 bp 175 bp Large fragment 376 bp Dde. I (a) Dde. I restriction sites in normal and sickle-cell alleles of -globin gene Dde. I restriction sites; in a normal person there Are two sites to be recognized; in a sickle cell Person, they are missing a site (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles After the gel is run, you can see that the sickle Cell is missing a band

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 This is how the “transfer” Is set up, to transfer the Bands from the gel onto A nylon membrane 60

Fig. 20 -11 a TECHNIQUE DNA + restriction enzyme Restriction fragments I II III Nitrocellulose membrane (blot) Heavy weight Gel Sponge I Normal -globin allele II Sickle-cell III Heterozygote allele 1 Preparation of restriction fragments Alkaline solution 2 Gel electrophoresis Paper towels 3 DNA transfer (blotting)

Fig. 20 -11 b Radioactively labeled probe for -globin gene I II III Step 4: the bag is sealed with a buffer and a Radioactive probe and sealed; this is placed In a hot water bath that gets agitated for a Certain amount of time; Once done a picture can be taken, counts Of the radiation can be taken and amounts of DNA can be figured 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

DNA Sequencing Relatively short DNA fragments can be sequenced by the dideoxy chain termination method Modified nucleotides called dideoxyribonucleotides (dd. NTP) attach to synthesized DNA strands of different lengths Each type of dd. NTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment The DNA sequence can be read from the 63 resulting spectrogram

Fig. 20 -12 a Single strand of DNA is put in microcentrifuge tube with primer, DNA polymerase, dd. NTP with Flourescence attached, and lose d. NTPs TECHNIQUE DNA (template strand) Primer Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) d. ATP d. CTP DNA polymerase dd. ATP dd. CTP d. TTP d. GTP dd. GTP https: //www. youtube. com/watch? v=lg. ASq. W bem. Cc

Fig. 20 -12 b TECHNIQUE DNA (template strand) Labeled strands Shortest Direction of movement of strands Longest labeled strand Detector Laser RESULTS Shortest labeled strand Last base of longest labeled strand Last base of shortest labeled strand All this mixed Together, any Time a dd. NTP is Added to the Replicating strands the strand stops elongating; eventually You have a bunch of Strands that can be Separated through a Gel, they will separate out by length, you can then put them in order using the flourescent Tag to determine the sequence

Analyzing Gene Expression Nucleic acid probes can hybridize with m. RNAs transcribed from a gene Probes can be used to identify where or when a gene is transcribed in an organism 66

Studying the Expression of Single Genes Changes in the expression of a gene during embryonic development can be tested using Northern blotting (looks at m. RNA) Reverse transcriptase-polymerase chain reaction Both methods are used to compare m. RNA from different developmental stages 67

Northern blotting combines gel electrophoresis of m. RNA followed by hybridization with a probe on a membrane Identification of m. RNA at a particular developmental stage suggests protein function at that stage Can also learn if there are differing amounts of the message in different tissue 68

Reverse transcriptase-polymerase chain reaction (RT-PCR) is quicker and more sensitive Reverse transcriptase is added to m. RNA to make c. DNA, which serves as a template for PCR amplification of the gene of interest The products are run on a gel and the m. RNA of interest identified 69

Fig. 20 -13 TECHNIQUE 1 c. DNA synthesis m. RNA, reverse transcriptase and all other necessary Components are mixed together and incubated c. DNAs 2 PCR amplification Primers added and goes through PCR Amplification to make more copies Primers -globin gene 3 Gel electrophoresis RESULTS Gel electrophoresis reveals the simplified DNA Made from the m. RNA Embryonic stages 1 2 3 4 5 6

In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific m. RNAs in place in the intact organism 71

Fig. 20 -14 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 compare patterns of gene expression in different tissues, at different times, or under different conditions https: //www. youtube. com/watch? v=VNs. Th MNj. Kh. M 73

Fig. 20 -15 TECHNIQUE 1 Isolate m. RNA. 2 Make c. DNA by reverse transcription, using fluorescently labeled nucleotides. 3 Apply the c. DNA mixture to a microarray, a different gene in each spot. The c. DNA hybridizes with any complementary DNA on the microarray. Tissue sample m. RNA molecules Labeled c. DNA molecules (single strands) DNA fragments representing specific genes DNA microarray 4 Rinse off excess c. DNA; 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 When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype 75

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 m. RNA https: //www. youtube. com/watch? v=2 d. L 7 Sh_ud. Ks Interference RNA 76

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 Organismal cloning: when a whole organism is cloned 77

Cloning Plants: Single-Cell Cultures One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organism A totipotent cell is one that can generate a complete new organism (these are able to de-differentiate!) Very common now, we do this through stems, leaves, roots of plants all the time! 78

Fig. 20 -16 EXPERIMENT RESULTS Transverse section of carrot root 2 -mg fragments Fragments were cultured in nutrient 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.

Cloning Animals: Nuclear Transplantation In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell Experiments with frog embryos have shown that a transplanted nucleus can often support normal development of the egg However, the older the donor nucleus (the one that gets transplanted into another cell), the lower the percentage of normally developing tadpoles 80

https: //www. youtube. com/watch? v=OCro 5 zfc 3 uc 81

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 82

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

Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent” CC is gray/white And playful While her mother “rainbow” is Calico and reserved 84

Fig. 20 -19 What are possible reasons for the differences Seen in CC compared to her mother? http: //www. youtube. com/watch? v=d. V 2 Ox. SGhwj. Y

Problems Associated with Animal Cloning 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 86

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 (pluripotent) 87

The adult body also has stem cells, which replace nonreproducing specialized cells John Gurdon, 79, of the Gurdon Institute in Cambridge, Britain and Shinya Yamanaka, 50, of Kyoto University in Japan, discovered ways to create tissue that would act like embryonic cells, without the need to collect the cells from embryos. Nobel Prize for Medicine awarded Oct 2012 88

Stem cells created from adult tissue are known as "induced pluripotency stem cells", or i. PS cells. Because patients may one day be treated with stem cells from their own tissue, their bodies might be less likely to reject them. 89

Fig. 20 -20 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

The aim of stem cell research is to supply cells for the repair of damaged or diseased organs Current research is being done to use stem cells for Parkinson’s, Alzheimer’s, Arthritis, hair loss? 91

Medical Applications One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases Not all diseases are genetic Some diseases involve changes in gene expression 92

Diagnosis of Diseases Scientists can diagnose many human genetic disorders by using PCR and primers corresponding to cloned disease genes, then sequencing the amplified product to look for the disease-causing mutation Genetic disorders can also be tested for using genetic markers that are linked to the disease-causing allele 93

PCR has been used to detect Sickle-cell anemia Hemophilia Cystic fibrosis Huntington’s disease Duchenne’s muscular dystrophy 94

Scientists can detect abnormal disease-causing alleles by testing for genetic markers they are very close to or linked to the allele that is being investigated Single nucleotide polymorphisms (SNPs) are useful genetic markers These are single base-pair sites that vary in a population 95

They can be coding or noncoding regions They are the basis of different alleles These differences are termed “polymorphisms” 96

Some SNPs alter the recognition sequence for a restriction enzyme When a restriction enzyme is added, SNPs result in DNA fragments with different lengths, or restriction fragment length polymorphism (RFLP) 97

Fig. 20 -21 The gene can be isolated then scientists can look for the SNP which is Linked to the abnormal Allele. You don’t need to sequence the entire gene Just look for the SNP DNA T Normal allele SNP C Disease-causing allele https: //www. youtube. com/watch? v=VJx. Imex 2 y. E

Human Gene Therapy Gene therapy is the alteration of an afflicted individual’s genes Gene therapy holds great potential for treating disorders traceable to a single defective gene Vectors are used for delivery of genes into specific types of cells, for example bone marrow Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in 99 future generations

Fig. 20 -22 Cloned gene 1 Insert RNA version of normal allele into retrovirus. Viral RNA 2 Retrovirus capsid Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. 3 Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient 4 Inject engineered cells into patient. Bone marrow

Pharmaceutical Products Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases 101

Synthesis of Small Molecules for Use as Drugs • The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor • Pharmaceutical products that are proteins can be synthesized on a large scale 102

Protein Production in Cell Cultures • Host cells in culture can be engineered to secrete a protein as it is made • This is useful for the production of insulin, human growth hormones, and vaccines 103

Protein Production by “Pharm” Animals and Plants Transgenic animals are made by introducing genes from one species into the genome of another animal Transgenic 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 https: //www. youtube. com/watch? v=Nmkj 5 gq 1 c. QU 104

Fig. 20 -23

Forensic Evidence and Genetic Profiles An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids Genetic profiles can be used to provide evidence in criminal and paternity cases and to identify human remains Genetic profiles can be analyzed using RFLP analysis by Southern blotting 106

Even more sensitive is the use of genetic markers called short tandem repeats (STRs), which are variations in the number of repeats of specific DNA sequences PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths The probability that two people who are not identical twins have the same STR markers is exceptionally small 107

WHO DID IT? 108

Look for the similarities in the banding pattern Suspect 1 is the offender 109

Fig. 20 -24 (a) This photo shows Earl Washington just before his release in 2001, after 17 years in prison. 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.

Environmental Cleanup Genetic engineering can be used to modify the metabolism of microorganisms Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials Biofuels make use of crops such as corn, soybeans, and cassava to replace fossil fuels 111

Agricultural Applications DNA technology is being used to improve agricultural productivity and food quality 112

Animal Husbandry Genetic engineering of transgenic animals speeds up the selective breeding process Beneficial genes can be transferred between varieties or species 113

Genetic Engineering in Plants Agricultural scientists have endowed a number of crop plants with genes for desirable traits The Ti plasmid is the most commonly used vector for introducing new genes into plant cells Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance, increased resistance to pests, increased resistance to salinity, and improved nutritional value of 114 crops

Fig. 20 -25 TECHNIQUE Agrobacterium tumefaciens Ti plasmid Site where restriction enzyme cuts T DNA with the gene of interest RESULTS Recombinant Ti plasmid Plant with new trait

Safety and Ethical Questions Raised by DNA Technology Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology 116

Most public concern about possible hazards centers on genetically modified (GM) organisms used as food Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives 117

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 118

You should now be able to: 1. Describe the natural function of restriction enzymes and explain how they are used in recombinant DNA technology 2. Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid 3. Define and distinguish between genomic libraries using plasmids, phages, and c. DNA 4. Describe the polymerase chain reaction (PCR) and explain the advantages and 119 limitations of this procedure

5. Explain how gel electrophoresis is used to analyze nucleic acids and to distinguish between two alleles of a gene 6. Describe and distinguish between the Southern blotting procedure, Northern blotting procedure, and RT-PCR 7. Distinguish between gene cloning, cell cloning, and organismal cloning 8. Describe how nuclear transplantation was used to produce Dolly, the first 120 cloned sheep

9. Describe the application of DNA technology to the diagnosis of genetic disease, the development of gene therapy, vaccine production, and the development of pharmaceutical products 10. Define a SNP and explain how it may produce a RFLP 11. Explain how DNA technology is used in the forensic sciences 121

12. Discuss the safety and ethical questions related to recombinant DNA studies and the biotechnology industry 122