Recipient Cell. DNA Foreign DNA. Recombinant DNA

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1 Module 4B Biotechnology In this module, we will examine some of the techniques scientists have developed to study and manipulate the DNA of living organisms. Objective # 7 Explain what genetic recombination is, why it is important, and how it occurs naturally. 1 2 Objective 7 Genetic recombination involves combining DNA from 2 different sources into a single molecule. Individual genes are not altered, they are simply joined together in new combinations. Genetic recombination is important because it produces new genetic types. 3 Objective 7 New genetic types are the raw material for evolution. As new genetic types are generated, they may gradually replace existing genetic types by the process of natural selection or by other evolutionary mechanisms. Thus, the rate of evolution depends directly on the rate at which new genetic types are generated. 4 Objective 7 In nature, combining DNA from 2 different individuals into a single molecule involves 2 steps: first, DNA from 2 individuals is combined in a single cell then DNA from both individuals is joined to form a single molecule Objective 7 In prokaryotes, several natural mechanisms can combine DNA from 2 different individuals into a single cell: Transformation a cell absorbs pieces of foreign DNA from its environment

2 Genetic Recombination by transformation: Recipient Cell 1 DNA Foreign DNA 2 Recombinant DNA 7 8 Objective 7 Plasmid uptake a cell absorbs plasmids from the environment. Transduction a virus acts as a vector to transfer pieces of foreign DNA from one cell to another. Conjugation a temporary cytoplasmic bridge connects 2 cells so that DNA can be passed from one cell to the other: 9 10 Objective 7 Once pieces of foreign DNA have entered a recipient cell, they often combine with the recipient cell s genome to form recombinant DNA. Plasmids, for example, can be integrated into, and excised from, specific locations on the main bacterial genome:

3 Objective 7 In prokaryotes, genetic recombination generally occurs by transferring pieces of foreign DNA into a recipient cell and then combining it with the recipient cell s genome. In most eukaryotes, recombination has become a regular part of the lifecycle. It occurs through fertilization followed by crossing over during meiosis: 13 Genetic Recombination in eukaryotes: Fertilization Crossing Over 14 Objective 7 In order to recombine DNA from 2 individuals through fertilization and crossing over, the 2 individuals must be able to mate with each other. Therefore they must belong to the same species. Objective # 8 Discuss the roles of restriction enzymes and DNA ligase in constructing artificially recombined DNA Objective 8 While various natural mechanisms can combine DNA from 2 individuals of the same species, scientists have developed techniques to combine DNA from any 2 individuals. These techniques result in the production of artificially recombined DNA. 17 Objective 8 Two key enzymes are used to make artificially recombined DNA. 1) Restriction enzymes (also called restriction endonucleases): cut DNA into fragments so called molecular scissors each one recognizes and cuts DNA only where a specific sequence of base pairs occurs. 18 3

4 Objective 8 many do not cut straight through both strands, but make a jagged cut leaving unpaired bases at both ends. Because these unpaired bases can pair with complimentary bases, they are called sticky ends. 2) DNA ligase is used to join DNA fragments together. This is the molecular glue Objective 8 Summary of procedure for making artificially recombined DNA: Isolate DNA from 2 different sources. Cut the DNA from both sources into fragments using the same restriction enzyme. Objective 8 Mix the DNA fragments together. Because they were cut with the same restriction enzyme, fragments from different sources will have the same sticky ends and can pair up. Use the enzyme DNA ligase to join the paired fragments together: Objective 8 Recombinant DNA technology can be used to create recombinant plasmids (or other recombinant agents such as viruses) which are useful for inserting foreign genes into recipient cells. Plasmids or other recombinant agents that are used to insert foreign DNA into recipient cells are called vectors:

5 Objective # 9 Describe how the following can be used to produce multiple copies of a DNA fragment: a) molecular cloning (gene cloning) b) polymerase chain reaction (PCR) Objective 9 Why would scientists want to produce multiple copies of a DNA fragment? to study its structure and function to compare the fragment with DNA from other sources if it codes for a useful protein, to produce large quantities of the protein Objective 9 There are 2 basic strategies for producing multiple copies of a gene: a) molecular cloning (gene cloning) b) polymerase chain reaction (PCR) Objective 9a a) With gene cloning, a vector is used to insert the gene we wish to clone into a host cell. The host cell then replicates the foreign gene using the same cellular machinery that it uses to replicate its own DNA Objective 9a During gene cloning, plasmids are often used as vectors to insert foreign genes into bacterial host cells. Using Ui plasmids with ih specific genetic traits can help scientists determine which bacterial cells have actually absorbed the gene we wish to clone:

6 31 Objective 9a Summary of procedure for gene cloning: Cut plasmids containing lac Z and amp resistance genes with a restriction enzyme. Use a restriction enzyme that cuts the plasmid once, inside the lac Z gene. Use the same restriction enzyme to cut DNA containing the gene you wish to clone. 32 Objective 9a Mix DNA from both sources together. Some plasmids will simply reclose. Other plasmids will join with a piece of foreign DNA to form a recombinant plasmid. Incubate bacterial cells with the plasmids. Some cells will absorb no plasmid, some will absorb a reclosed plasmid, and some will absorb a recombinant plasmid. 33 Objective 9a When plated on media containing ampicillin and X-gal, how do we know which bacterial cells absorbed no plasmid? These cells will not survive because they lack the gene for ampicillin resistance. Therefore no colonies are formed. 34 Objective 9a When plated on media containing ampicillin and X-gal, how do we know which bacterial cells absorbed a reclosed (non-recombinant) plasmid? These cells have a functional lac-z gene. Therefore they will make the enzyme β-galactosidase and will form blue colonies. 35 Objective 9a When plated on media containing ampicillin and X-gal, how do we know which bacterial cells absorbed a recombinant plasmid? The inserted foreign DNA will inactivate the lac-z gene. Therefore these cells do not make β-galactosidase and will form white colonies. 36 6

7 Objective 9a How do we know which white colonies contain the specific gene of interest? The white colonies can be screened for the specific gene of interest using a genetic probe. A genetic probe is a radioactive molecule of RNA or single-stranded stranded DNA that is complementary to the gene of interest. 37 Using a Genetic Probe to Screen for the Gene of Interest 1. Colonies of bacteria, each grown from cells taken from a white colony. 2. A replica of the plate is made by pressing a filter against the colonies. Some cells from each colony adhere to the filter. Filter 3. The filter is washed with a solution that denatures the DNA and contains the radioactively labeled probe. The probe contains nucleotide sequences complementary to the gene of interest and binds to cells containing the gene. 5. A comparison with the original plate identifies the colony containing the gene. Film Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 4. Only those colonies containing the gene will retain the probe and emit radioactivity on film placed over the filter. 38 Objective 9b b) A second method for producing multiple copies of a gene is PCR With PCR, we create the conditions needed for DNA replication inside a test tube that contains a copy of the gene: Objective 9b Summary of procedure for polymerase chain reaction (PCR): 1) Denaturation a solution containing RNA primers and the DNA fragment to be amplified is heated so that the DNA dissociates into single strands

8 Objective 9b Objective 9b 2) Annealing of primers the solution is cooled, and the primers bind to complementary sequences on the DNA flanking the gene to be amplified. 3) Primer extension DNA polymerase then copies the remainder of each strand, beginning at the primer. 43 Repeat steps 1 3 many times, each time doubling the number of copies, until a sufficient number of copies are produced. 44 Objective # 10 Objective 10 Explain the difference between the following types of DNA libraries: a) Genomic libraries b) cdna libraries A DNA library is a collection of DNA fragments representing all the DNA of an organism Objective 10a The simplest kind of DNA library is a genomic library. To create a genomic library, the entire genome of an organism is fragmented. The fragments are then inserted into a vector, such as a plasmid or phage, and introduced into a host: Plasmid Library DNA fragments from source DNA DNA inserted into plasmid vector Transformation Phage Library DNA fragments from source DNA DNA inserted into phage vector Phages infect E. coli 47 Each cell contains a single fragment. All cells together are the library. Each phage contains a single fragment. All phage together are the library. Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 48 8

9 Objective 10b Another type of DNA library is a cdna library. A cdna library includes only DNA fragments that actually code for proteins rather than all DNA fragments. This means that introns and other non-coding sections of the genome are not included. Objective 10b To produce a cdna library, scientists first isolate the mature mrna from an organism. An enzyme called reverse transcriptase is then used to make a complementary DNA copy of each mature mrna molecule: Objective 10b If you want to make bacterial cells that can manufacture a particular human protein, why is it important to insert cdna rather than the original genomic DNA into the bacterial cells? Objective # 11 Explain how a DNA fragment containing a particular nucleotide sequence can be isolated and identified from a sample containing many different DNA fragments. Objective 11 A A DNA fragment containing a particular nucleotide sequence can be isolated and identified from a sample containing many different DNA fragments using a procedure developed by E.M. Southern called the Southern blot procedure:

10 Objective # 12 Explain the process and importance of RFLP analysis and DNA fingerprinting Objective 12 As we have seen, restriction enzymes can be used to cut DNA into fragments called restriction fragments. However, these cuts are not made at random, each restriction enzyme cuts the DNA only where a particular sequence of bases occurs. These are called recognition sites. 57 Objective 12 If DNA from 2 individuals is different, then the location of recognition sites for a particular restriction enzyme may also be different. If we cut DNA from both individuals with the same restriction enzyme, we may get different size fragments. This is called a restriction fragment length polymorphism (RFLP). 58 Objective 12 How can we determine the length of the fragments that are produced when we treat DNA from 2 individuals with the same restriction enzyme? Gel Electrophoresis

11 Objective 12 RFLP analysis is a powerful technique that is being in the field of forensics: small amounts of DNA collected at a crime scene can be amplified using PCR the DNA is cut into fragments with a restriction enzyme, and the fragments are separated using gel electrophoresis Objective 12 the resulting banding pattern is then compared with the banding pattern produced by DNA samples from different suspects the banding pattern for each individual is essentially unique, and is referred to as a DNA fingerprint Objective 12 Objective 12 RFLPs can also be used to distinguish between DNA that contains different alleles if different recognition sites occur within or very close to the different alleles. This is referred to as genetic screening:

12 Objective # 13 Explain the process and importance of DNA sequencing. Objective 13 DNA sequencing involves determining the actual sequence of base pairs in a DNA molecule. This is the ultimate level of genetic analysis. A method of sequencing called enzymatic sequencing was developed by Fredrick Sanger Objective 13 Sanger s method uses modified nucleotides called dideoxynucleotides. Dideoxynucleotides have an H in place of an OH at both the 2 position and the 3 position of the sugar. As a result, if a dideoxynucleotide is incorporated into a growing nucleotide chain, no additional nucleotides can be added and chain elongation is terminated. Dideoxynucleotides have an H in place of an OH at both the 2 position and the 3 position of the sugar. O O P O CH 2 5 O O N N 1 NH 2 N 69 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. H H Objective # 14 Describe at least 4 ways genetic technology can be used for human benefit

13 1) Introduce genes coding for proteins with commercial or medical value into other organisms, such as bacteria, in order to mass produce the proteins. Proteins produced in this way include: Human insulin helps regulate blood sugar level Interferons assist the immune response by inhibiting viral replication 73 Human growth hormone stimulates cell division and growth Erythropoietin stimulates red blood cell production Atrial peptides may be a new way to treat high blood pressure and kidney failure Tissue plasminogen activator (TPA) dissolves blood clots 74 2) Produce vaccines that provide protection against disease. Genetic technology has been used to develop two types of vaccines : subunit vaccines and DNA vaccines. A subunit vaccine is developed using a small portion (or subunit) of the pathogen - for example, a protein in the coat or envelope that surrounds a harmful virus. 75 To prepare a subunit vaccine against a harmful virus, a gene coding for a protein in the coat or envelope of the harmful virus is spliced into the genome of a harmless virus like vaccinia. 76 Next, the modified vaccinia virus, which contains surface proteins from the harmful virus, is injected into uninfected people. The immune system detects the proteins from the harmful virus on the surface of vaccinia and makes antibodies against any virus with those proteins. 77 Construction of a subunit vaccine against herpes simplex: Herpes simplex virus Human immune response 6. Antibodies directed against herpes simplex viral coat are made. 1. DNA is extracted. Harmless vaccinia (cowpox) virus Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2. Herpes simplex gene is isolated. 3. Vaccinia DNA is extracted and cleaved. 5. Harmless engineered virus (the vaccine) with surface like herpes simplex is injected into the human body. Gene specifying herpes simplex surface protein 4. Fragment containing surface gene combines with cleaved vaccinia DNA

14 To prepare a DNA vaccine, a gene from a pathogen is artificially replicated and then injected directly into uninfected people. If human cells take up the gene, some may use it to make the protein encoded by the gene. The presence of the foreign protein in the body triggers an immune response against the pathogen. 79 Unlike subunit vaccines, DNA vaccines do not stimulate the production of antibodies against the pathogen. Instead, they stimulate the activity of killer T-cells, which are another component of the body s immune response ) Alter the human genome to cure genetic disease or give people certain desirable traits. Many yg genetic disorders are caused by a single defective allele. In gene therapy, scientists try to supply a copy of the normal allele to those cells that need it but lack it There are some serious obstacles to successful gene therapy: How do you get a copy of the gene into enough of the cells that need it? Will the gene function normally once it is inserted into a cell? Will inserting a new gene into a cell damage or alter the expression of any other genes? 84 14

15 During gene therapy, there is always the concern that insertion of a normal allele into a cell could inactivate another essential gene or turn on a gene inappropriately. Gene therapy was first used successfully to treat SCID (severe combined immunodeficiency). The procedure involved removing white blood cells from the patient, using a virus to insert the necessary gene into the cells, and then returning the cells to the bloodstream. Although the treatment was successful, about 15% of patients developed a rare form of leukemia Scientists determined that the vector used to introduce the normal allele into white blood cells integrated into the genome next to a proto-oncogene oncogene called LM02. Activation of this gene caused the leukemias. 4) Genetically alter organisms, including crops and livestock, to give them certain desirable traits such as disease resistance, frost resistance, faster growth rate, or higher nutritional value. Organisms that have genes introduced without the use of conventional breeding are called transgenic: Transgenic Rice Beans Aspergillus fungus Wild rice Daffodil Ferritin gene is transferred into rice from beans. Phytase gene is transferred into rice from a fungus. Metallothionin gene is transferred into rice from wild rice. Enzymes for -carotene synthesis are transferred into rice from daffodils. Rice Fe Pt chromosome S A 1 A 2 A 3 A 4 Ferritin protein increases iron content of rice. Phytate, which Metallothionin -carotene, a inhibits iron protein supplies precursor to reabsorption, extra sulfur to vitamin A, is is destroyed by the increase iron synthesized. phytase enzyme. uptake. Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

16 Objective # 15 Discuss some potential problems associated with genetic technology. Objective 15 1) Some question the safety of eating genetically modified organisms. So far, no negative effects have been documented. 2) Some worry that genes from genetically modified organisms may spread into the gene pools of wild organisms and modify them. There is no evidence this has occurred Objective 15 3) Another concern is that genetically altered organisms may escape into the environment and replace natural organisms or upset the balance of nature. 4) There are also moral and ethical questions associated with controlling the genetic make-up and evolution of existing life forms, including humans

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