Objective # 7 Module 4B – Biotechnology In this module, we will examine Explain what genetic some of the techniques scientists recombination is, why it is have developed to study and important, and how it occu rs manipulate the DNA of living naturally. organisms. 1 2 Objective 7 Objective 7 Genetic recombination involves New genetic types are the raw material combining DNA from 2 different for evolution. As new genetic types are sources into a single molecule. generated, they may gradually replace Individual genes are not altered, existing genetic types by the process of thiljidhihey are simply joined together in natur a l se lec tion or by o the r evo lu tiona ry new combinations. mechanisms. Genetic recombination is Thus, the rate of evolution depends important because it produces new directly on the rate at which new genetic genetic types. types are generated. 3 4 Objective 7 Objective 7 In nature, combining DNA from 2 In prokaryotes, several natural different individuals into a single mechanisms can combine DNA from molecule involves 2 steps: 2 different individuals into a single cell: first, DNA from 2 individuals is Transformation – a cell absorbs pieces combined in a single cell of foreign DNA from its environment. then DNA from both individuals is joined to form a single molecule 5 6 1 Genetic Recombination by transformation: Recipient Cell 1 DNA 2 Foreign DNA 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 f orm reco mbinan t DNA. Plasmids, for example, can be integrated into, and excised from, specific locations on the main bacterial genome: 11 12 2 Objective 7 Genetic Recombination in eukaryotes: In prokaryotes, genetic recombination generally occurs by transferring pieces Fertilization 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 Crossing Over by crossing over during meiosis: 13 14 Objective 7 Objective # 8 In order to recombine DNA from 2 Discuss the roles of restriction individuals through fertilization and crossing over, the 2 individuals must enzymes and DNA ligase in bbltmtithhthrbe able to mate with each other. constructing artificially recombined Therefore they must belong to the DNA. same species. 15 16 Objective 8 Objective 8 Two key enzymes are used to make While various natural mechanisms can combine DNA from 2 individuals of the artificially recombined DNA. same species, scientists have developed 1) Restriction enzymes (also called techniqqyues to combine DNA from any 2 restriction endonucleases): individuals. cut DNA into fragments – so called These techniques result in the “molecular scissors” production of artificially recombined each one recognizes and cuts DNA DNADNA.. only where a specific sequence of 17 base pairs occurs. 18 3 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 ligaseis used to join DNA fragments together. This is the “molecular glue”. 19 20 Objective 8 Objective 8 .Summary of procedure for making Mix the DNA fragments together. artificially recombined DNA: Because they were cut with the same restriction enzyme, fragments from Isolate DNA from 2 different sources. different sources will have the same “sticky ends” and can pair up. Cut the DNA from both sources into fragments using the same restriction Use the enzyme DNA ligase to join enzyme. the paired fragments together: 21 22 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::vectors 23 24 4 Objective # 9 Objective 9 Describe how the following can be Why would scientists want to produce used to produce multiple copies of a multiple copies of a DNA fragment? DNA fragment: to study its structure and function a) molecular cloninggg (gene cloning ) to compa re t he frag me nt w it h DN A b) polymerase chain reaction (PCR) from other sources if it codes for a useful protein, to produce large quantities of the protein 25 26 Objective 9 Objective 9a There are 2 basic strategies for producing a) With gene cloning, a vector is used to multiple copies of a gene: insert the gene we wish to clone into a host cell. The host cell then a) molecular cloning (gene cloning) replicates the foreign gene using the b) polhii(PCR)lymerase chain reaction (PCR) same cellular machinery that it uses to replicate its own DNA. 27 28 Objective 9a During gene cloning, plasmids are often used as vectors to insert foreign genes into bacterial host cells. UiUsing p lasm ids w ihith spec ific genet ic traits can help scientists determine which bacterial cells have actually absorbed the gene we wish to clone: 29 30 5 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 31 clone. 32 Objective 9a Objective 9a Mix DNA from both sources together. When plated on media containing Some plasmids will simply reclose. Other ampicillin and XX--gal,gal, how do we know plasmids will join with a piece of foreign which bacterial cells absorbed no DNA to form a recombinant plasmid. plasmid? Incubate bacterial cells with the plasmids. These cells will not survive because Some cells will absorb no plasmid, some they lack the gene for ampicillin will absorb a reclosed plasmid, and some resistance. Therefore no colonies are will absorb a recombinant plasmid. formed. 33 34 Objective 9a Objective 9a When plated on media containing When plated on media containing ampicillin and XX--gal,gal, how do we know ampicillin and XX--gal,gal, how do we know which bacterial cells absorbed a which bacterial cells absorbed a reclosed (non(non--recombinant)recombinant) plasmid? recombinant plasmid? These cells have a functional lac-lac-ZZ The inserted foreign DNA will gene. Therefore they will make the inactivate the lac-lac-ZZ gene. Therefore enzyme β--galactosidasegalactosidase and will form these cells do not make β--galactosidasegalactosidase blue colonies. and will form white colonies. 35 36 6 Objective 9a Using a Genetic Probe to Screen for the Gene of Interest How do we know which white 1. Colonies of bacteria, each grown 5. A comparison with the original from cells taken from a white colony. plate identifies the colony colonies contain the specific gene of containing the gene. 2. A replica of the plate is made by pressing a filter against the interest? colonies. Some cells from each colony adhere to the filter. The white colonies can be screened for Filter Film the specific gene of interest using a 3. The filter is washed with a solution 4. Only those colonies genetic probeprobe.. A geneticgenetic probe is a that denatures the DNA and contains containing the gene will the radioactively labeled probe. The retain the probe and emit probe contains nucleotide sequences radioactivity on film placed radioactive molecule of RNA or complementary to the gene of interest over the filter. and binds to cells containing the gene. singlesingle--strandedstranded DNA that is complementary to the gene of interest. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 37 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: 39 40 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. 41 42 7 Objective 9b Objective 9b 2) Annealing of primers – the solution Repeat steps 1 – 3 many times, each is cooled, and the primers bind to time doubling the number of copies, complementary sequences on the until a sufficient number of copies are DNA flanking the gene to be produced. amplified. 3) Primer extension – DNA polymerase then copies the remainder of each strand, beginning at the primer. 43 44 Objective # 10 Objective 10 Explain the difference between the following types of DNA A DNA library is a collection of DNA libraries: fragments representing all the DNA of a) Genomic libraries an orimrganism. b) cDNA libraries 45 46 Objective 10a Plasmid Library Phage Library DNA fragments DNA fragments from source DNA from source DNA The simplest kind of DNA library is a genomic librarylibrary.. DNA inserted DNA inserted into plasmid vector into phage vector To create a genomic library, the entire ggggenome of an organism is fragmented. The fragments are then inserted into a Transformation Phages infect E. coli vector, such as a plasmid or phage, and introduced into a host: Each cell contains a Each phage contains a single fragment. All cells single fragment. All phage together are the library.