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Quizzes - PGM

Quizzes - PGM. 2007. Quiz 1 – 26 September 2007. Please print, use ink, and avoid teensy-weensy printing. Name any one of 2 methods for getting 500 ug of a human gene of your choice other than growing up millions of flasks of cells. PCR, cloning

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Quizzes - PGM

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  1. Quizzes - PGM 2007

  2. Quiz 1 – 26 September 2007 Please print, use ink, and avoid teensy-weensy printing. • Name any one of 2 methods for getting 500 ug of a human gene of your choice other than growing up millions of flasks of cells. • PCR, cloning • True or False. The first homework assignment is due on Friday of this week. • True • The process of making an RNA copy from a DNA template is called transcription. • The process of making a replica of DNA is called _replication_____________. • Usually there are two copies of any given gene in a mammalian cell. Explain. • One copy on each of two homologous chromosomes

  3. Quiz 2 – 28 September 2007 Remember to put last name first on the cover and the page on which you are writing your answer. Please use pen and avoid teensy weensy lettering. • Give any 2 reasons why plasmid and bacteriophage DNAs are practical for getting enough of YFG. • Check lecture notes. • To work in silico, what piece of equipment do you need? Computer • True or False. The purpose of the methylase in a restriction system is to make it possible for restriction endonucleases to cut foreign DNA. • True, if you think of methylase as protecting the genomic DNA so foreign DNA is cut. • False, if you thought the question meant that addition of a methyl to a site makes that specific site easier to cut. • True or False. An RE that recognizes 5’CAATTG3’ also recognizes 5’GTTAAC3’. • False

  4. Quiz 3 – 1 October 2007 • Name two types of ends produced by the various Type II restriction enzymes. • Blunt and sticky • Which sequence of nucleotides would you predict would appear more frequently in any genome? A) GACGTC or B) ACGT • What type of enzyme might a genetic engineer use to protect a specific DNA site from restriction enzyme digestion? A DNA methylase specific for the RE and its recognition sequence. • What method is used to separate RE digest fragments from each other on the basis of size? • Gel electrophoresis, most usually agarose. • Explain why the results of a restriction endonuclease digest are predictable. • The recognition sequence for a given RE is known, so it’s position in the sequence of any specific source of DNA will always be the same.

  5. Quiz 4 – 3 October 2007 1. True or False: The restriction enzyme recognition sequences that appear in a useful multiple cloning site do not appear elsewhere in the plasmid. True 2,3. There are two major “pieces” of DNA that you need to prepare in the process of making a recombinant DNA construct. What are they? Vector and Insert 4,5. Name the enzyme used for: • First strand synthesis of cDNA Reverse transcriptase (RT) • Nicking of RNA that is base paired with first strand synthesis cDNA. RNAse H • 2nd strand synthesis of cDNA. DNA polymerase I (or sometimes RT) • Making the ends of ds cDNA blunt. T4 DNA polymerase, with both polymerase and 3’ exonuclease activity. Last Name, First Name 1,2. There are two major “pieces” of DNA that you need to prepare in the process of making a recombinant DNA construct. What are they? 3,4. Name the enzyme used for: • A) First strand synthesis of cDNA • B) Nicking of RNA that is base paired with first strand synthesis cDNA. • C) 2nd strand synthesis of cDNA. • D) Making the ends of ds cDNA blunt. 5. True or False: The restriction enzyme recognition sequences that appear in a useful multiple cloning site do not appear elsewhere in the plasmid.

  6. Quiz 5 – 5 October 2007 • What is one way of making the ends of a polished ds cDNA compatible with a vector that has sticky ends? Add adaptors with ends for the same RE recognition sequence as at the vector sticky ends. • True or False. Sticky ends can be ligated even when no 5’ phosphate is present. • What is the name for the process of introducing naked DNA into bacterial cells? Transformation. Transfection, transduction, infection, and conjugation will be clarified next time. • If you are making a plasmid cDNA clone, and you have completed the process referred to in Question 3, what is the purpose of spreading the bacterial cells onto a plate containing the drug ampicillin? To determine whether plasmid vector has been taken up successfully. Drug resistance is now rarely used to reveal whether there is insert, and only works if the vector has two different drug resistance genes. • What is the name of the enzyme that breaks down a lactose analog resulting in a blue color? LacZ or beta-galactosidase.

  7. Quiz 6 – 8 October 2007 • If your strategy is to place a cDNA insert into the Bam HI site of a vector A) What adaptors do you use to give your cDNA sticky ends? a) Pst I b) Bam HI c) you don’t need adaptors B) What restriction enzyme do you use to get the insert out of your construct after you have amplified and purified enough of your construct to work with? Bam HI • Only some plasmid vectors allow you to determine the presence or absence of an insert using X-gal-based blue/white selection. Explain in one sentence.Only vectors that includes the gene for LacZ’ (including at least one interior cloning site) can be used for blue/white discrimination. • Only some bacterial strains allow you to determine the presence or absence of an insert using X-gal-based blue/white selection. Explain in one sentence. Only strains that contain a gene for the alpha domain of beta-galactosidase can be used. (These bacteria must also be negative for the complete beta-gal gene, so that complementation by a plasmid-coded LacZ’ can be detected.) • Consider the following RE recognition site: AGC/GCT Show the one and only one Class II cut site that would produce blunt ends.

  8. In the absence of ligase, sticky ends are sticky because of A) covalent or B) non-covalent bonds The phosphodiester bond created by ligase is A) covalent or B) non-covalent In a blot hybridization protocol, what is the word for the labeled DNA that includes your sequence of interest? Probe What is the word given to blot hybridization in which RNA has been blotted to the membrane from a gel? Northern If you had only the figure of the Hind III digest shown at right, and knew nothing about the starting DNA, which map(s) on the chalk board might be accurate? A only A and B A, B, and C It was all three, but you had to be there. Quiz 7 – 10 October 2007

  9. Quiz 8 – 12 October 2007 • In which phosphate do you want your radioactive label to be if you are going to label a DNA probe by the random priming method? Alpha Beta Gamma • (True or False) Producing a labeled cRNA probe requires a primer. False • New DNA is synthesized during: A) labeling by chemical cross-linking B) labeling by random priming C) both A and B • To label the 5’ end of a strand of DNA you will need: A) polymerase B) kinase, (and maybe phosphatase first) C) both 5) Which end of a DNA strand is labeled by terminal deoxynucleotidyl transferase (TdT)? 3’ end

  10. Quiz 9 – 15 October 2007 • How is light produced in all the non-radioactive visualization methods we discussed in class? By an enzyme. • Name one way in which the location of light produced by non-radioactive probes is visualized. Autoradiography or digital capture or phosphorimaging • Name one way in which the location of radioactive probes on a membrane is visualized. Autoradiography or phosphorimaging • What is the difference between direct and indirect non-radioactive labeling procedures? Direct: the light producing enzyme is covalently linked to the probe nucleic acid chain. Indirect: The enzyme is not covalently bound to the probe. The light producing enzyme is covalently attached to a binding molecule which is in turn allowed to bind non-covalently to the small molecule covalently bound to the probe. • If you are probing for digest fragments from YFG, what sequence must be present in the probe you use? Sequence found somewhere in YFG.

  11. Quiz 10 – 17 October 2007 • Name one of the two major advantages of using bacteriophage instead of conventional plasmids for construction of libraries. • Phage will get DNA into a greater % of bacteria in the culture. • Phage can carry larger inserts of DNA. • Phage can get larger pieces of DNA into bacteria more efficiently. • Name any two of the 3 required parts of the lambda phage genome that are required for the lytic life cycle. • Lambda left arm • Lambda right arm • Cos sites • What is the name given to a “colony” of phage on an agar plate? Plaque – also accepted “clone” • Why would you want a genomic library to have larger inserts than a cDNA library? So that your inserts could include as much of your entire gene as possible. The gene is much larger than the mRNA because of the presence of introns and the importance of flanking sequences, Also accepted: the larger the inserts, the fewer clones. But this answer really addresses the question: Why would you want to use a vector that can accommodate the largest possible inserts? That answer is not really relevant when comparing a genomic library to a cDNA library, because cDNA clone number is dictated by the prevalence of different RNA types, and insert size is dictated by the length of the mRNA. T or F. A cDNA library made from the mRNA from one type of cell will be the same as a cDNA library made from any other type of cell. False Different cell types make different mRNAs – that’s what makes them different.

  12. Quiz 11 – 19 October 2007 • T or F – In order to remove the stuffer (central region) from a lambda vector, you need to cut the vector at the COS sites. False • The average appearance of a restriction site for a 4-hitter in any sequence is once every 250 bp. The insert size for a genomic library in a lambda vector is typically about 20kb. However, inserts for the library are frequently prepared with a 4-hitter. Explain. A partial digest with a 4-hitter allows you to select the size range you want. • If two lambda left or two lambda right arms ligate to each other during preparation of a genomic library, the product will not be packaged into phage. Explain. This question was not graded. In the case of two lambda rights, the product would definitely be too short for packaging. The same may also be true for 2 lambda lefts. Even if packaged, the result would be non-productive because genes required for phage replication are missing. • What is a potential advantage of treating your prepared genomic insert fragments with phosphatase? They won’t ligate to each other and be removed from making inserts in phage lengths that can be packaged. • T or F. After ligation of inserts to arms, a lambda library is transformed into competent bacteria. No, it must be packaged and then allowed to infect or transduce.

  13. Quiz 12 – 22 November 2007 • When using a lambda vector, what step must occur after ligation and before introduction of DNA into the host bacterial cells? In vitro packaging • How does a genomic library serve to separate your DNA of interest from all the rest of the DNA in the genome? Each smaller region of DNA is in a separate clone. • Name a method that is used to find the interest you want in a genomic library. Plaque or colony hybridization. • What feature of any good vector allows you to grow up enough of the clone you want? High copy number (or independent replication, although it’s really a combination of independent replication to high copy number). • What would be the starting material for making the inserts of a chimpanzee genomic library? Chimp DNA(that is, total high quality DNA, and not chimp mRNA)

  14. Quiz 13 – 24 October 2007 • What is the purpose of the equation at right? The equation was the one for determining the number of clones that must be plated out in order to have a “complete” library. • How is F in the equation at right determined when making a genomic library? F is determined on the basis of the average size of the fragments you will be using as inserts. Average insert size is numerator. Size of the genome with which you are working is the denominator. • T or F. The template for making cDNA is genomic DNA. False. It is RNA. • Would you be happier if your plated library contained mostly blue plaques or mostly clear plaques? Mostly clear placques, because that means most of your plaques have inserts disrupting the lac Z’ gene. (In the case of plaques, the blue color is generated within the bacteria before they have lysed.) 5. Is the translation start site in an exon or an intron? It is in an exon, because the translation start site must be present in the mRNA, and the mRNA includes only sequence from the exons in the gene.

  15. Quiz 14 (-2) – Halloween, 2007 • Name any one high capacity vector other than a cosmid. P1, PAC, BAC, YAC • Use one or two sentences to describe any one feature of a cosmid that contributes to its name. Cosmids are plasmids that include cos sites, which allow for packaging and efficient transfer of DNA into host cells during the library construction phase. The constructs are concatomerized before packaging. Once inside the cell, the linear ds DNA circularizes by annealing at the cos sites, and then the construct replicates like a plasmid because it has a plasmid Ori but none of the genes required for the lambda life cycle. There is also a drug selection gene to maintain bacteria that carry the plasmid. • What substrate used in chain termination sequencing causes the chain termination? The dideoxynucleotides • How are the four bases distinguished in current fluorescence-based sequencing techniques? Each base of the 4 ddNTPs is labeled with a different color. • What is it about the cycle sequencing process that minimizes the amount of template necessary? The same template is used repeatedly by cycling through denaturation after each synthesis step, and then allowing a new primer to anneal so that synthesis can begin again. This means you need fewer templates to achieve the amount of product you need to be able to detect fluorescence for each of the products of different length.

  16. Quiz 15 (-2) – 2 November 2007 • Name either one of 2 currently used massively parallel sequencing methods. Solexa(Illumina) or 454 • Name any one major difference between the outcomes of Solexa (or 454) sequencing and conventional cycle sequencing. Light or fluorescence is produced with addition of each base; all molecules are continuously synthesized, with no permanent termination. • Name any one required reagent for PCR. Template, dNTPs, heat stable DNA polymerase, 2 opposing primers. • Name any one way in which PCR differs from fluorescence-based cycle sequencing. PCR uses 2 opposing primers; sequencing uses only 1 primer. No ddNTPs are used in PCR. PCR doubles logarithmically; sequencing increases the number of fragments linearly. Final PCR products are all the same length; final cycle sequencing products must differ in length to be able to read the sequence. • Name any one application of PCR. Please check the lecture. There are also correct answers that were not listed in the lecture notes.

  17. Quiz 16 (-2) – 5 November 2007 • What is the “trick” used in screening a large library that reduces the total number of PCR reactions required? (One word, starts with “p”) Pooling clones • What does STR stand for? short tandem repeat • What must be true about the number of STRs in the population at a single locus in order for the locus to be useful for identification purposes? Number must be highly variable. • What is the “trick” by which PCR can be used to subclone between two different restriction sites? Design the restriction sites you want into your primers. • Name any one way in which the products of a PCR reaction can be visualized. Gel electrophoresis and staining; fluorescent labeling of the primers and capillary electrophoresis with laser scanner; real time PCR with intercalated fluorescent dye

  18. Quiz 17 (-2) 9 November 2007 • Describe in one or two sentences the in silico method of determining potentially important DNA sequences in the regulatory region of a gene. Align sequences across species and look for conserved regions. • In EMSA, what 2 types of molecules interact to cause the mobility shift? DNA and protein. • What organism do you use to grow up enough reporter construct for a reporter assay in mouse cells? E. coli. • You compare the results from two luciferase reporter constructs, -1000 to +1, and -900 to +1. You record more light with the longer construct than with the shorter. What do you conclude? (2 points) The region from -1000 to -900 contains sequence that functions in some way to enhance transcription.

  19. Quiz 18 – 14 November 2007bonus points • Both ChIP and EMSA study the interactions of DNA with what type of molecule? Protein • Which of the two methods, ChIP or EMSA, traps interactions in vivo? ChIP • Which of the two methods uses a reversible covalent crosslink in the process? ChIP • Name two different ways in which the DNA of interest in the ChIP assay is identified? PCR and direct sequencing and microarray hybridization • Who would be more likely to use ChIP and EMSA, someone interested in transcription or someone interested in translation?

  20. Quiz 19 – 16 November 2007 • Name any two of the 3 features all good cloning plasmids have in common. Ori for independent replication, drug resistance, multiple cloning site, etc. • Which type of end prevents Exonuclease III digestion – a) 5’ overhang or b) 5’ recessed? • What distinguishes any reporter plasmid from other cloning plasmids? Includes a gene that is expressed to report the activity of the promoter that is being studied. • What distinguishes any expression plasmid from other cloning plasmids? Includes a transcription cassette. • In what organism do you grow up enough of your cloning plasmid to work with? E. coli

  21. Quiz 20 – 19 November 2007 • There are 4 general approaches to changing an expression system to solve various problems that may come up. For example, one of them is to change the sequence or structure of the gene being expressed. The general approaches can be used singly, or in combination. NAME ANY 1 OF THE OTHER 3 APPROACHES. Change the transcription cassette; modify the host cell; change to a different host cell (which may require changing the expression construct). • Who are the other members of your powerpoint team? • Briefly list what you yourself (just you) have done so far to contribute to the power point assignment. • Do you think your team members would agree that your answer to number 3 is accurate? • Briefly list what the other members of the team have already contributed.

  22. Quiz 20 – 21 November 2007 • In what organism is an expression plasmid that does not contain YFgene amplified so that you have enough to work with? E.coli • In what organism is an expression plasmid into which you have inserted YFgene amplified so that you have enough to work with? E.coli • Which resistance gene is used to select for stable incorporation of YF Expression Construct into a mouse cell expression system? Ampicillin or Neo/G418 • Name any 4 ways in which DNA can be transferred into eukaryotic cells. Calcium phosphate and DEAE dextran precipitation, dendrimers, lipid or liposome-mediated, viral transfer, bacterial (plant), electroporation, biolistics, microinjection. • Name any one thing for which you are thankful. Many wonderful answers.

  23. Quiz 21 (18 total for denominator -2 because drop 2 lowest = 16) 28 November 2007 • The following two questions refer to the procedure for making knockout mice that we described in class. 1. What is it that serves to knock out YF Gene in the procedure for making knockout mice? The G418 resistance gene. 2. True or False – The TK gene will be present in a knockout mouse made by the procedure described in class. • The following 3 questions refer to finding a disease gene. 3. Name any one polymorphic phenotype that can be used as a marker to help locate the region of the genome in which a “disease gene” lies. STRs, VNTRs, SNPs, RFLPs. Be sure you know what these letters stand for and what each really is. 4. True or False – The polymorphism in a marker is usually what causes the disease. 5. What method could you use to show that the gene you have found is actually the gene that, when mutated, causes the disease of interest? Check the last several slides of Monday’s lecture. (There can be more than one answer. Use no more than 3 sentences.)

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