GENE 301 1st EditionExam # 3 Study Guide LecturesDefinitions:Recombinant DNA: Bacterial plasmid DNA joined to human DNATaq: Enzyme similar to E. coli DNA polymerase-I but capable of withstanding boiling temperaturesEnzyme used as a DNA polymerase in the PCR techniquePhotolyase: One type of DNA repair of thymine dimmers, that requires no DNA synthesis (enzyme)FISH: Method used to determine the location of a DNA segment on chromosomesA fluorescent dye labeled primer that is known to hybridize to human DNA at only one site in the genome is used to prime human DNA in chromosomes on microscope slide for the PCR reaction. A four base pair sequence (GACA) is used as an unlabeled return primer for this PCR reaction. After 17 thermal cycles the slide is observed with a fluorescence microscope. What is this process called?Dideoxythymidine: Compound used in preparing a sequencing gel only for the DNA run in the “T” laneSouthern blot: Technique used to transfer the DNA from an electrophoretic gel to a (nitrocellulose) membraneWalking DNA: Process that uses segments of one DNA library to identify adjacent segments in another DNA library. Nitro-cellulose: Absorbs the single stranded DNA eluted from a DNA electrophoresis gel in a “Southern Blot”RNA polymerase: Enzyme that binds to the promoter site2,3-dideoxyadenosine: Used to terminate synthesis in one column (of the 4 columns) of a DNA sequencing gelPolymerase III: Enzyme modified by recA so that it can function as a polymerase in SOS repairTranslation: Silencing or interfering RNA stops this processes hnRNA: Enzyme used to amplify the effects of interfering RNA.siRNA: Used to stop translation of a specific message in eukaryotes.The translation of a message can be halted and the message marked for degradation by which of the following? siRNAIn eukaryotes, which of the following can serve to stop translation AND prevent further translation of a messenger RNA? siRNAThe polymerase chain reaction was run on whole human DNA using 16 different 8 base pair, random sequence primers. The resulting DNA fragments were separated by electrophoresis on a gel. There were 60 bands on the resulting. What is this process called? RAPDTo insert a segment of Drosophila DNA into a bacterial plasmid, one needs Eco-R1 restriction endonuclease and what other enzyme? LigaseThe polymerase chain reaction amplifies specific sequences of DNA. What best describes the DNA that isamplified exponentially? The DNA between the labeled primer and the return primer. What is the purpose of ‘walking’ two different libraries of DNA fragments?To determine the order of sequences in both libraries.FISH is a PCR technique that uses a DNA template the DNA obtained from:A chromosome smearWhat would be the effect on the operation of the arginine operon (negative, repressible) if the regulator protein were mutated so that it could not bind to arginine?Constitutive synthesis A single copy of siRNA can result in the destruction of multiple copies of message. What enzyme (newly discovered in eukaryotes) is responsible for amplifying the effect of one siRNA?RDRP (RNA dependent RNA polymerase)A region of DNA in a particular cell synthesizes a segment of RNA that is 174 bases long and in the form of a large palindrome (shRNA). The RNA is transported to the cytoplasm and folds into a hairpin loop of double stranded RNA. In the cytoplasm, the hairpin loop is recognized by a double-stranded RNA cutting enzyme (dicer) and is cut into 22 BP lengths. When these 22 BP double stranded segments are combinedwith protein, they function by:Splicing the introns out of messenger RNA.A 28 base pair probe is available that binds to only one site in a haploid human genome. This probe is fluorescently labeled and is used as a primer for PCR amplification. A secondary unlabeled primer with the sequence GGAGA is used. The primers are combined with whole human DNA, Taq, and the 4 nucleotides, and then the PCR reaction is cycled 20 times. What would be your estimate of the average size of the fluorescently labeled segment(s) that will be amplified?45=1024 bpAssuming that the individual from which the whole DNA was taken (from above) is homozygous for all regions involved, how many different fluorescently labeled bands are expected to be on the gel from the above experiment?1If you wanted to produce 50-100 different DNA segments by PCR to make a DNA ‘fingerprint’ of mitochondrial DNA (17,000 bp long), what length of primer would you add (using the same primer for both directions (SPAR)) to accomplish this?4bpA certain strain of E. coli is constitutive for the enzymes for lactose metabolism. On an F’ plasmid you have the choice of adding one (but only one) element of the lac system to make the bacteria a merodiploid for that element. Assume the original E. coli has wild-type structural loci. If your goal is to determine whether the lac operon is constitutive because of a mutant regulator OR because of a mutant operator, what one wild-type element of the lac operon would you add using sexduction?Regulator geneAssume the mutant above is a constitutive operator. What phenotype would result if both a wild-type operator and a wild-type promoter (but no other elements) were added to the F’ plasmid?Constitutive DNA Gel QuestionsThe top is 5X because it is made of larger fragments and does not move as quickly down the gel. So B is an example of a homozygous 5X. A is an example of homozygous 4X and E is an example of homozygous 3X because it is the smallest fragment it moves the largest distance. C is heterozygous for 4X and 3X. D is heterozygous for 5X and 3X and F is heterozygous for 5X and 4X. E is the correct answer to this question. To do question 2 you need to use the formula [a]=q=aa+12Aatotal so14 +12(20+19)100=.335For this start at the bottom of the gel in the picture. That is the 5’ so it’ll be TATATGCATCACA. Now you have to flip it so that it goes 3’ATATACGTAGTGTThe correct answer is f. 3Determine the phenotype with respect to beta-galactosidase (z), permease (y) and transacetylase (a) of each combination as U=uninducible, I= inducible, and C= constitutive.a. i+p+o+z+y-a+/i-p+o+z-y-a+ (IUI)b. iSp+o+z+y-a+/i+p+ocz-y+a+ (UCC)c. For this you have to map it out and physically draw it. __________6 __________1 ___________5 ___________3 ____________4 _____________2It’ll look similar to this. The answers are B and CThe answer is B. It’s done just like the one above