Quiz Answers

PGM 2012

Quiz 1 – 26 September 2012Quiz 1 – 26 September 20121,2.1,2.PCR and cloning have enabled molecular biologists PCR and cloning have enabled molecular biologists

to overcome 3 major hurdles that can stand in the to overcome 3 major hurdles that can stand in the way of working with a favorite gene. Name any two way of working with a favorite gene. Name any two of those hurdles. of those hurdles. get enough of the DNA in a cost-effective manner;get enough of the DNA in a cost-effective manner;identify the gene of interest;identify the gene of interest;get the gene of interest away from all the rest of the get the gene of interest away from all the rest of the DNA in the genome from which it comesDNA in the genome from which it comes

3.3. T or T or FF. Being “diploid” and being “double-stranded” . Being “diploid” and being “double-stranded” are the same thing.are the same thing.

4.4. T or T or FF. The fruit fly is interesting evolutionarily, but . The fruit fly is interesting evolutionarily, but has no parallels in human gene studies.has no parallels in human gene studies.

5.5. T T or F. Chromatin that is tightly packed and not or F. Chromatin that is tightly packed and not being transcribed is called heterochromatin.being transcribed is called heterochromatin.

Quiz 2 – 1 October 2012Quiz 2 – 1 October 20121.1. Name the type of enzyme that modifies bacterial genomic Name the type of enzyme that modifies bacterial genomic

DNA so that it isn’t cut by its own RE.DNA so that it isn’t cut by its own RE.Restriction methylaseRestriction methylase

2.2. TRUE OR TRUE OR FALSEFALSE. An 8-hitter will likely have more cut sites . An 8-hitter will likely have more cut sites in any given piece of DNA than a 6-hitter will.in any given piece of DNA than a 6-hitter will.

3.3. TRUE OR TRUE OR FALSEFALSE. The enzymes that recognize. The enzymes that recognize5’ G/AATTC 3’ AND 5’ C/AATTG 3’ are 5’ G/AATTC 3’ AND 5’ C/AATTG 3’ are

isoschizomers.isoschizomers.4.4. A diagram that indicates where on a piece of DNA various A diagram that indicates where on a piece of DNA various

REs cut is called a restriction _REs cut is called a restriction _MAPMAP________.________.5.5. TRUE OR TRUE OR FALSEFALSE. Gs and Cs occur more frequently in . Gs and Cs occur more frequently in

mammalian DNA than As and Ts do.mammalian DNA than As and Ts do.

6.6. M.S. students only – please tell me in whose lab you M.S. students only – please tell me in whose lab you are/will be doing your MS research.are/will be doing your MS research.

Quiz 3 – 3 October 2012Quiz 3 – 3 October 20121)1) Answer one:Answer one:

PFGE stands for __PFGE stands for __Pulsed Field Gel ElectrophoresisPulsed Field Gel Electrophoresis____________Or OFAGE stands for _Or OFAGE stands for _Orthogonal Field Agarose Gel ElectrophoresisOrthogonal Field Agarose Gel Electrophoresis

2)2) OFAGE separates DNA both on the basis of size and on the basis of how OFAGE separates DNA both on the basis of size and on the basis of how long it takes the molecules to _long it takes the molecules to _reorientreorient____________. (This can be ____________. (This can be answered with one word, but use more if you need to.)answered with one word, but use more if you need to.)

3)3) You have decided that you would like to make a cDNA clone of your You have decided that you would like to make a cDNA clone of your favorite mRNA. What is the first thing that you do?favorite mRNA. What is the first thing that you do?Make a plan.Make a plan.

4)4) The abbreviation for the origin of replication in a plasmid is The abbreviation for the origin of replication in a plasmid is ORIORI..

5)5) Explain why amplification of YFG as part of a plasmid is said to occur “in Explain why amplification of YFG as part of a plasmid is said to occur “in vivo”. vivo”. The actual amplification takes place within a living organism, the The actual amplification takes place within a living organism, the bacterial host.bacterial host.

Quiz 4 – 8 October 2012Quiz 4 – 8 October 20121)1) Name the enzyme that is used to polish or blunt any overhanging ends of a double Name the enzyme that is used to polish or blunt any overhanging ends of a double

strand cDNA. strand cDNA. T4 DNA polymeraseT4 DNA polymerase2)2) Name the enzyme that is used to make covalent bonds between vector, in our Name the enzyme that is used to make covalent bonds between vector, in our

case pGEM3Z, and insert. case pGEM3Z, and insert. DNA ligaseDNA ligase3)3) WhatWhat is the name of the process for introducing “naked” DNA into is the name of the process for introducing “naked” DNA into competentcompetent

bacterial cells? bacterial cells? TransformationTransformation4)4) You complete the steps described in #2 and #3. You then plate the bacteria. You You complete the steps described in #2 and #3. You then plate the bacteria. You

are careful to plate onto agar that contains ampicillin. This is important because:are careful to plate onto agar that contains ampicillin. This is important because:a) bacteria need ampicillin in order to growa) bacteria need ampicillin in order to growb) only bacteria that have taken up the construct you want will growb) only bacteria that have taken up the construct you want will growc) only bacteria that have taken up vector, either with or without an insert, will c) only bacteria that have taken up vector, either with or without an insert, will grow.grow.

5)5) You look at the colonies that grew as a result of #4 above. They are all white. You look at the colonies that grew as a result of #4 above. They are all white. Give at least two different explanations for why you have all white colonies on the Give at least two different explanations for why you have all white colonies on the plate. plate. 1) you didn’t add X-gal to the plate.1) you didn’t add X-gal to the plate.2) either the bacterial strain or the plasmid you were using did not contain the 2) either the bacterial strain or the plasmid you were using did not contain the gene for the appropriate beta-galactosidase domain, or contained an unknown gene for the appropriate beta-galactosidase domain, or contained an unknown mutation such that the correct protein was not produced.mutation such that the correct protein was not produced.3) your construction steps went fabulously well, and the plasmids in all your 3) your construction steps went fabulously well, and the plasmids in all your colonies had inserts.colonies had inserts.

5)5) T or T or FF. You can use exonuclease digestion followed by agarose gel . You can use exonuclease digestion followed by agarose gel electrophoresis to determine the approximate length of insert in a construct.electrophoresis to determine the approximate length of insert in a construct.

Quiz 6 – 15 October 2012Quiz 6 – 15 October 20121.1. Name one method of “uniform” labeling of a probe. Name one method of “uniform” labeling of a probe. Cross-Cross-

linking, random priming, cRNA synthesis.linking, random priming, cRNA synthesis.2.2. What are the two substrates for polynucleotide kinase in What are the two substrates for polynucleotide kinase in

the 5’ end labeling reaction? the 5’ end labeling reaction? Gamma labeled ATP, free 5’ Gamma labeled ATP, free 5’ hydroxyl on a nucleotide.hydroxyl on a nucleotide.

3.3. Name any one method of probe labeling that results in the Name any one method of probe labeling that results in the synthesis of new nucleic acid. synthesis of new nucleic acid. 3’ end-filling, TdT 3- end 3’ end-filling, TdT 3- end labeling, cRNA synthesislabeling, cRNA synthesis

4.4. Which of the following is non-covalent?Which of the following is non-covalent?a.a. Bonding of biotin to DNA probe.Bonding of biotin to DNA probe.b.b. Bonding of streptavidin to biotin.Bonding of streptavidin to biotin.c.c. Bonding of alkaline phosphatase to streptavidinBonding of alkaline phosphatase to streptavidin

5.5. Name one method by which the light produced by alkaline Name one method by which the light produced by alkaline phosphatase or horseradish peroxidase can be visualized.phosphatase or horseradish peroxidase can be visualized.phosphorimaging, digital camera imaging, phosphorimaging, digital camera imaging, autoradiographyautoradiography

Quiz 7 – 22 October 2012Quiz 7 – 22 October 20121.1. T T or F. Viral transduction can introduce DNA into a higher percentage or F. Viral transduction can introduce DNA into a higher percentage

of an appropriate culture of E. coli cells than standard (chemical) of an appropriate culture of E. coli cells than standard (chemical) transformation can.transformation can.

2.2. Define “genomic” library. Define “genomic” library. A collection of clones that together contain A collection of clones that together contain inserts representing all the DNA in cells of a particular organism.inserts representing all the DNA in cells of a particular organism.

3.3. When preparing DNA inserts for a genomic library, you need to make When preparing DNA inserts for a genomic library, you need to make sure that the fragments meet three criteria. Name any two.sure that the fragments meet three criteria. Name any two.correct size for the vector of choice, regions of overlapping sequence correct size for the vector of choice, regions of overlapping sequence among fragments, ends compatible with the vector.among fragments, ends compatible with the vector.

1.1. In order to prepare a concatomer for packaging in lambda, you must In order to prepare a concatomer for packaging in lambda, you must perform which of the following in vitro:perform which of the following in vitro:a) rolling circle replication a) rolling circle replication b) ligationb) ligation

5.5. When using a replacement lambda vector, what 3 DNA sections or When using a replacement lambda vector, what 3 DNA sections or regions must be found between cos sites in order for successful regions must be found between cos sites in order for successful packaging to occur? packaging to occur? Lambda left, lambda right, insertLambda left, lambda right, insert

6.6. Successful appearance of plaques on a plate depend upon Successful appearance of plaques on a plate depend upon successful completion of which lambda life cycle?successful completion of which lambda life cycle?a) lytica) lytic b) lysogenic b) lysogenic

Quiz 8 – 31 October 2012Quiz 8 – 31 October 20121.1. Name any two of the 3 Name any two of the 3 sequencesequence features that expression plasmids features that expression plasmids

and basic cloning plasmids have in common. and basic cloning plasmids have in common. Ori, DrugOri, DrugRR gene, gene, cloning sitecloning site

2.2. What is the most important difference between an expression plasmid What is the most important difference between an expression plasmid and a basic cloning plasmid?and a basic cloning plasmid?Expression plasmid is used to express a protein from an expression Expression plasmid is used to express a protein from an expression cassette. Basic cloning plasmid does not contain appropriate cassette. Basic cloning plasmid does not contain appropriate sequences for expression of YFG(cDNA).sequences for expression of YFG(cDNA).

1.1. What is the difference between a constitutive promoter and an What is the difference between a constitutive promoter and an inducible promoter? inducible promoter? Constitutive promoter is always on. Inducible is Constitutive promoter is always on. Inducible is off unless a chemical is supplied in the medium. The chemical acts to off unless a chemical is supplied in the medium. The chemical acts to activate the promoter.activate the promoter.

2.2. T or T or FF. “Fusion protein” is the term applied to proteins that consist of . “Fusion protein” is the term applied to proteins that consist of two different subunits that are non-covalently bonded to each other.two different subunits that are non-covalently bonded to each other.

3.3. T or T or FF. Tomorrow is Halloween.. Tomorrow is Halloween.4.4. A) Who are your teammates for the term project?A) Who are your teammates for the term project?

B) Has your team identified someone to “interview”?B) Has your team identified someone to “interview”?

Quiz 9 – 7 November 2012 (open book)Quiz 9 – 7 November 2012 (open book)1.1. Name any 2 transfection methods for getting a DNA construct into eukaryotic cells. Name any 2 transfection methods for getting a DNA construct into eukaryotic cells.

electroporation, precipitation, charged polymer, lipid-mediated, biolistics, microinjection.electroporation, precipitation, charged polymer, lipid-mediated, biolistics, microinjection.

2.2. TT or F. Packaging of DNA into eukaryotic viral particles is done in cultured eukaryotic or F. Packaging of DNA into eukaryotic viral particles is done in cultured eukaryotic cells.cells.

3.3. Name any one advantage of using virally mediated transfer of DNA into eukaryotic Name any one advantage of using virally mediated transfer of DNA into eukaryotic cells.cells.

A greater % of intended expressor cells will effectively take up DNA.A greater % of intended expressor cells will effectively take up DNA.

When the DNA integrates, it is likely to integrate in a more predictable way than When the DNA integrates, it is likely to integrate in a more predictable way than transfected DNA is. transfected DNA is.

Others understood the question differently and responded that eukaryotic cells are able Others understood the question differently and responded that eukaryotic cells are able to perform post-translational modification appropriately (given that YFG is eukaryotic).to perform post-translational modification appropriately (given that YFG is eukaryotic).

4.4. Name any one way in which the process for stable transfection differs from the process Name any one way in which the process for stable transfection differs from the process for transient transfection.for transient transfection.

A selection method is usually required. Inserted DNA is heritable.A selection method is usually required. Inserted DNA is heritable.

5.5. Put your sketch of your HDAC/GFP expression construct into your bluebook when you Put your sketch of your HDAC/GFP expression construct into your bluebook when you pass it forward (or sketch it into your bluebook).pass it forward (or sketch it into your bluebook).

Minimum correct answer required continguous promoter and a fusion protein region of Minimum correct answer required continguous promoter and a fusion protein region of HDAC/GFP. Class discussion amplified on this.HDAC/GFP. Class discussion amplified on this.

Quiz 10 – 19 November 2012

1. T or F. All three methods of sequencing we have discussed depend upon synthesis of a complementary DNA strand.

2. T or F. All three methods of sequencing we have discussed depend upon the use of nucleotides that are modified to block further synthesis.

3. Name the method of choice for determining the sequence of a 1 kb insert of a plasmid clone.

Dideoxy or Sanger-Coulson sequencing

4. The genetic distance between two loci is based on recombination__________ frequency______________.

5. T or F. Markers good for positional cloning of a disease gene are genetically linked to the disease gene.