Genetics Lab Exam 1
Genetics Lab Exam 1 Bio 184
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This 6 page Study Guide was uploaded by kaylee goff on Tuesday September 20, 2016. The Study Guide belongs to Bio 184 at California State University Sacramento taught by Thomas Peavy in Fall 2016. Since its upload, it has received 7 views. For similar materials see General Genetics in Biology at California State University Sacramento.
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Date Created: 09/20/16
Genetics study guide Vocab Recombinant DNA technology is a set of tools that allows molecular biologists to create novel DNA molecules that do not exist in nature, and to produce large quantities of such “recombinant DNAs” for research and therapeutic purposes. -The basic process by which recombinant DNA molecules are made is quite simple. First, it is important to have a DNA molecule called a vector that can be manipulated in vitro so that a foreign piece of DNA can be inserted into the vector. The recombinant plasmid can then be introduced into a living organism (usually bacteria) to replicate it along with its own DNA and then studied. In some cases, the protein encoded by the foreign DNA within the vector is the desired product (e.g. humulin) and can be isolated from the bacterial host in large quantities by puriﬁcation processes. However, in our case, the desired outcome is to recover the recombinant plasmid from the bacterial host so the foreign insert can be DNA sequenced to determine the identity of the DNA. Vector: a DNA molecule used as a vehicle to artiﬁcially carry foreign genetic material into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artiﬁcial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker. Restriction enzyme cleavage sites: allow for the insertion of a transgene insert sticky ends: an end of a DNA double helix at which a few unpaired nucleotides of one strand extend beyond the other. 5'-ATCTGACT ______+ GATGCGTATGCT-3' 3'-TAGACTGACTACG________CATACGA-5' blunt ends: The end of a DNA fragment resulting from the breaking of DNA molecule in which there are no unpaired bases, hence, both strands are of the same length. Origin of replication (Ori): DNA sequence ensures that the plasmid will be recognized by the bacterial replication machinery and replicated along with the bacterial chromosome. The plasmid remains an autonomous unit and generally replicates to very high copy number within a single bacterial cell (1,000 or more copies/cell). selectable marker: allows only those cells that actually pick up the plasmid to be selected for against the background of all the cells that did not. The most common selectable markers are antibiotic resistance genes. multiple cloning site: Restriction enzymes will only cut DNA at speciﬁc sites. For example, the restriction enzyme “Eco RI” cuts DNA only at the sequence: 5’-GAATTC-3’. (Note the complementary strand will have the same sequence with the opposite polarity.) A multiple cloning site contains the sequences for several different restriction enzymes, giving the researcher ﬂexibility in his/her experimental strategy. Plasmids: small double-stranded circles of DNA, which are naturally carried by some bacteria in addition to their larger circular genome. -In nature, plasmids carry extra information not normally required for survival of the cell, such as genes for antibiotic resistance or toxic proteins that enable the bacterium to better invade its host. Restriction enzymes: DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonuclease -In this experiment we use two restriction enzymes(EcoRI and NsiI) Ligase: an enzyme that can catalyze the joining of two large molecules by forming a new chemical bond, usually with accompanying hydrolysis of a small pendant chemical group on one of the larger molecules or the enzyme catalyzing the linking together of two compounds transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material (exogenous DNA) from its surroundings through the cell membrane(s). Transformation occurs naturally in some species of bacteria 16s ribosomal RNA gene: The foreign DNA we are interested in sequencing, that is common to all bacteria which can be used to determine the taxonomic identity of the organism. -Our role in this project is to “clone” the 16s rRNA gene fragment that was PCR ampliﬁed by students in our Biology department as part of their Bio 2 laboratory course. The context of the word “clone” in this case means to insert the PCR fragment into a vector and recover this recombinant plasmid from a colony of bacteria within which all their DNA is identical Plasmid is inserted into a vector, the vector is then introduced to a living organism (E.coli) in hopes of transforming the bacterial cell. The transformed cell will take up the plasmid DNA and will then replicate (aka clone) the plasmid DNA, and that will be studied. -The context of the word “clone” in this case means to insert the PCR fragment into a vector and recover this recombinant plasmid from a colony of bacteria within which all their DNA is identical. • Why do bacteria make restriction enzymes and how are they used in this experiment? Restriction enzyme, also called restriction endonuclease, a protein produced by bacteria that cleaves DNA at speciﬁc sites along the molecule. In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating infecting organisms. -Each student will isolate a recombinant plasmid DNA from a unique bacterial colony and subsequently analyze it by enzymatically digesting the DNA using two different restriction enzymes (EcoRI and NsiI) • Describe the major components of a vector. The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. All vectors may be used for cloning and are therefore cloning vectors -**** not E. coli -The manipulation of DNA is normally conducted on E. coli -Insertion of a vector into the target cell is usually called transformation for bacterial cells • What are the advantages to using E.coli as the host organism for rDNA technology? Escherichia coli because it is easy to culture and is not usually harmful to humans making it an ideal organism for growing up recombinant DNA molecules. It is also easily grown through asexual cycles in as little as 20 minutes, generally has just one gene per trait, can have colonies representing over 100 individuals displayed on a single Petri plate, can participate in gene exchange by conjugation, transformation, and transduction, and serves as host to numerous viruses and plasmids. • What equipment was used and explain how and why they are useful. The kit we’ll be using is the GeneJET Plasmid MiniPrep system from Thermo Scientiﬁc. Traps the plasmid DNA. • Know the controls for the described experiments. Know why they were used and if the results would change if the controls were not used. U undigested control: added EcoRI buffer , water L+ control: added "scDNA" which is a supercoiled plasmid that contains the kanamycin resistant gene. L - control: to the competent L cell tube, we added 2 uL of sterile water to make it a negative control. • After you performed the ligation reaction, what types of products were produced? Be able to draw and label them. Which type(s) are selected after transformed and plated on kanamycin agar plates, which would be selected if plated on an ampicillin agar plate. ampicilian will kill the E.coli cells • Know all reagents, solutions and DNA used for each experiment. Why is each important, can a reagent be deleted if so, would it change the results? If yes, then how, if no then why not. RNase A: a protein used To remove the RNA. -Pancreatic ribonuclease (RNase) is an endoribonuclease. It catalyzes the cleavage of the phosphodiester bond between the 5’-ribose of a nucleotide and the phosphate group attached to the 3’-ribose of an adjacent pyrimidine nucleotide. -allows us to isolate DNA for the transformation. -Resuspension Solution contains the RNase A -Lysis Solution to break open the cells and release their contents. The Lysis Solution contains SDS, a negatively-charged detergent, to solubilize the cell membranes and denature cellular proteins. The Lysis Solution is also basic (high pH), which denatures DNA (both chromosomal and plasmid). As the cells lyse, the large circular chromosome is partially fragmented into large, linear pieces. The plasmid DNA, being much smaller, remains intact. When the Lysis Solution is added, the large linear pieces separate from one another (lose their partner strands) but the plasmid DNA strands remain unbroken and together, like links in a chain -In the next step, a Neutralization Solution is added. This solution causes the DNA to renature (become double-stranded again). The chromosomal fragments get trapped in the protein debris as they attempt to ﬁnd their partner strands but the plasmid DNA zips back together quickly. When the solution is centrifuged, the chromosomal DNA joins the proteins and other cell debris in the pellet, while the plasmid DNA remains soluble. This is the basis for the separation of the chromosomal DNA from the plasmid DNA and is critical to the success of the technique. -Elution Buffer (the last step of the GeneJet procedure) removes chaotropic salts from the neutralization solution left behind in the GeneJet column. -The Neutralization Solution contains chaotropic salts that dehydrate the plasmid DNA and promote its binding to the GeneJET spin column. • Describe sticky ends and blunt ends. How do they differ, why would you chose one over another? sticky ends: an end of a DNA double helix at which a few unpaired nucleotides of one strand extend beyond the other. 5'-ATCTGACT ______+ GATGCGTATGCT-3' 3’-TAGACTGACTACG________CATACGA-5’ sticky ends cause a loss of DNA because one side of the strand is missing bases. blunt ends: The end of a DNA fragment resulting from the breaking of DNA molecule in which there are no unpaired bases, hence, both strands are of the same length. • The restriction enzyme NotI recognizes an 8 bp (palindromic) sequence. On average, how often would this enzyme cut a random sequence of DNA? How many times would the enzyme cut the E. coli genome? (The E. coli genome contains 4.6 x 106 bp.) fragments will occur ~every 65,000 bp • What is the difference between agar and agarose? When did you use each of these during these experiments? Agar is a heterogeneous mixture of two classes of polysaccharide: agaropectin and agarose. Although both polysaccharide classes share the same galactose-based backbone, agaropectin is heavily modiﬁed with acidic side-groups, such as sulfate and pyruvate. The neutral charge and lower degree of chemical complexity of agarose make it less likely to interact with biomolecules, and, therefore, agarose has become the preferred matrix for work with proteins and nucleic acids. -Agar was used to plate the bacteria and grow the colonies. Agarose was used in the gel electrophoresis portion of the experiment. • Be able to explain how agarose gel electrophoresis works. Describe how a gel rig is assembled, making and running the gel, and observing the bands. Very small volumes (~12 uL) of DNA samples are placed in wells at one end of the gel. The gel is submerged in a buffer, which carries the electric charge and dissipates heat around the gel. When an electric charge is applied, the molecules migrate through the gel based on size and charge. Since nucleic acids have a net negative charge from their phosphate groups and a uniform negative charge distribution along the entire molecule, they will move toward the positive electrode based primarily on size. Larger molecules will be impeded more by the gel's pores and will thus migrate more slowly. Circular molecules of the same molecular weight (i. e., same number of nucleotides) may migrate at different rates if they are supercoiled (twisted; imagine “winding-up” a rubber band by holding one end and rolling the other end between your thumb and ﬁngers) or relaxed (somewhat uncoiled due to a break in one of the two strands). Plasmids with a tight concentration tend to move through the gel more rapidly and easily than those with loose conformations that get “hung up” as they attempt to move through the pores. -When the gel is removed from the electric ﬁeld (the electrophoresis chamber), the molecules will be present in a lane extending from the sample well, but they will not be visible. One way to make nucleic acids visible is to treat the gel with an intercalating dye that ﬂuoresces when exposed to ultraviolet (U.V.) light. When a dye like SyberSafe is used, the DNA molecules show up as bright ﬂuorescent bands crossing the lanes. -DNA samples are loaded into the wells sequentially using a micropipette. (5-6). When the power supply is turned on, the DNA samples move into the gel toward the positive end and separate from one another according to size. During the actual gel run, the DNA fragments will not be visible, but they can be seen after SyberSafe staining of the gel when exposed to UV light. • Why are DNAsamples loaded at the negative end of a gel apparatus? An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge. The movement of charged molecules is called migration. Molecules migrate towards the opposite charge. A molecule with a negative charge will therefore be pulled towards the positive end (opposites attract!). DNA is negatively charged, so it is placed on the negative side, so that it is drawn to the positive side and migrates across the gel. • Describe the DNAstandard, why was it used, why was it heated. A molecular-weight size marker, also referred to as a protein ladder, DNA ladder, or RNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis. It is used as a comparison or control for the other unknown DNA gels. It works based on the principle that molecular weight is inversely proportional to migration rate through a gel. • Know why the same plasmid, from two different plasmid preps, may run differently on a gel (faster or slower). Different conformations. Plasmid DNA can exist in three conformations: supercoiled, open- circular (oc), and linear (supercoiled plasmid DNA is often referred to as covalently closed circular DNA, ccc). supercoiled DNA runs faster than open-circular DNA. Linear DNA runs through a gel end ﬁrst and thus sustains less friction than open-circular DNA, but more than supercoiled • Be able to analyze a gel and identify what the bands represent. Study the photograph on page 18 of your lab manual very carefully and know what you are seeing in each lane. • After a ligation of a vector and insert, be able to draw the multiple possible ligation products (monomers and multimers). After each of these products are purified by a plasmid prep, be able to distinguish between these different ligation products using different restriction enzyme digests and running them on a gel. 1. A bacterial plasmid is manipulated with enzymes outside the cell in a test tube. First, a double stranded break in the plasmid DNA molecule is made using a restriction enzyme. Next, the gene of interest (e.g. 16s rRNA) is introduced to the test tube, along with an enzyme called ligase that seals the DNA molecule back together. The resulting molecule is called a recombinant plasmid. 2.The recombinant plasmid is introduced into E. coli cells by transformation. Some bacterial cells take up plasmids naturally, but E. coli must be “coaxed” to do so. There are several methods for making the cells “competent” to take up plasmids. In this lab, we will treat the cells with calcium chloride, followed by a brief exposure to elevated temperature (“heat shock”). 3.As the transformed E. coli cells replicate, the plasmid is also replicated and can produce recombinant protein. Thus, a large amount of the desired recombinant DNA and/or expressed protein (e.g. humulin) is produced as the E. coli culture grows (doubling time ∼20min). 4.The cells are lysed open and the plasmid DNA or protein of interest is puriﬁed. *plasmids are very negatively-charged (like all DNA)
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