Quiz Answers. PGM 2012. Quiz 1 – 26 September 2012. 1,2. PCR and cloning have enabled molecular biologists to overcome 3 major hurdles that can stand in the way of working with a favorite gene. Name any two of those hurdles. get enough of the DNA in a cost-effective manner;
1,2. PCR and cloning have enabled molecular biologists to overcome 3 major hurdles that can stand in the way of working with a favorite gene. Name any two of those hurdles.
get enough of the DNA in a cost-effective manner;
identify the gene of interest;
get the gene of interest away from all the rest of the DNA in the genome from which it comes
5. T or F. Chromatin that is tightly packed and not being transcribed is called heterochromatin.
Name the type of enzyme that modifies bacterial genomic DNA so that it isn’t cut by its own RE.
2. TRUE OR FALSE. An 8-hitter will likely have more cut sites in any given piece of DNA than a 6-hitter will.
3. TRUE OR FALSE. The enzymes that recognize
5’ G/AATTC 3’ AND 5’ C/AATTG 3’ are isoschizomers.
4. A diagram that indicates where on a piece of DNA various REs cut is called a restriction _MAP________.
5. TRUE OR FALSE. Gs and Cs occur more frequently in mammalian DNA than As and Ts do.
6. M.S. students only – please tell me in whose lab you are/will be doing your MS research.
1) Answer one:
PFGE stands for __Pulsed Field Gel Electrophoresis______
Or OFAGE stands for _Orthogonal Field Agarose Gel Electrophoresis
OFAGE separates DNA both on the basis of size and on the basis of how long it takes the molecules to _reorient____________. (This can be answered with one word, but use more if you need to.)
You have decided that you would like to make a cDNA clone of your favorite mRNA. What is the first thing that you do?
Make a plan.
The abbreviation for the origin of replication in a plasmid is ORI.
5) Explain why amplification of YFG as part of a plasmid is said to occur “in vivo”. The actual amplification takes place within a living organism, the bacterial host.
Name the enzyme that is used to polish or blunt any overhanging ends of a double strand cDNA. T4 DNA polymerase
Name the enzyme that is used to make covalent bonds between vector, in our case pGEM3Z, and insert. DNA ligase
What is the name of the process for introducing “naked” DNA into competent bacterial cells? Transformation
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:
a) bacteria need ampicillin in order to grow
b) only bacteria that have taken up the construct you want will grow
c) only bacteria that have taken up vector, either with or without an insert, will grow.
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 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 gene for the appropriate beta-galactosidase domain, or contained an unknown mutation such that the correct protein was not produced.
3) your construction steps went fabulously well, and the plasmids in all your colonies had inserts.
T or F. You can use exonuclease digestion followed by agarose gel electrophoresis to determine the approximate length of insert in a construct.
Name one method of “uniform” labeling of a probe. Cross-linking, random priming, cRNA synthesis.
What are the two substrates for polynucleotide kinase in the 5’ end labeling reaction? Gamma labeled ATP, free 5’ hydroxyl on a nucleotide.
Name any one method of probe labeling that results in the synthesis of new nucleic acid. 3’ end-filling, TdT 3- end labeling, cRNA synthesis
Which of the following is non-covalent?
Bonding of biotin to DNA probe.
Bonding of streptavidin to biotin.
Bonding of alkaline phosphatase to streptavidin
Name one method by which the light produced by alkaline phosphatase or horseradish peroxidase can be visualized.
phosphorimaging, digital camera imaging, autoradiography
T or F. Viral transduction can introduce DNA into a higher percentage of an appropriate culture of E. coli cells than standard (chemical) transformation can.
Define “genomic” library. A collection of clones that together contain inserts representing all the DNA in cells of a particular organism.
When preparing DNA inserts for a genomic library, you need to make sure that the fragments meet three criteria. Name any two.
correct size for the vector of choice, regions of overlapping sequence among fragments, ends compatible with the vector.
In order to prepare a concatomer for packaging in lambda, you must perform which of the following in vitro:
a) rolling circle replication b) ligation
When using a replacement lambda vector, what 3 DNA sections or regions must be found between cos sites in order for successful packaging to occur? Lambda left, lambda right, insert
Successful appearance of plaques on a plate depend upon successful completion of which lambda life cycle?
a) lytic b) lysogenic
Name any two of the 3 sequence features that expression plasmids and basic cloning plasmids have in common. Ori, DrugR gene, cloning site
What is the most important difference between an expression plasmid and a basic cloning plasmid?
Expression plasmid is used to express a protein from an expression cassette. Basic cloning plasmid does not contain appropriate sequences for expression of YFG(cDNA).
What is the difference between a constitutive promoter and an inducible promoter? Constitutive promoter is always on. Inducible is off unless a chemical is supplied in the medium. The chemical acts to activate the promoter.
T or F. “Fusion protein” is the term applied to proteins that consist of two different subunits that are non-covalently bonded to each other.
T or F. Tomorrow is Halloween.
A) Who are your teammates for the term project?
B) Has your team identified someone to “interview”?
Name any 2 transfection methods for getting a DNA construct into eukaryotic cells.
electroporation, precipitation, charged polymer, lipid-mediated, biolistics, microinjection.
2.Tor F. Packaging of DNA into eukaryotic viral particles is done in cultured eukaryotic cells.
3. Name any one advantage of using virally mediated transfer of DNA into eukaryotic cells.
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 transfected DNA is.
Others understood the question differently and responded that eukaryotic cells are able to perform post-translational modification appropriately (given that YFG is eukaryotic).
4. Name any one way in which the process for stable transfection differs from the process for transient transfection.
A selection method is usually required. Inserted DNA is heritable.
Put your sketch of your HDAC/GFP expression construct into your bluebook when you pass it forward (or sketch it into your bluebook).
Minimum correct answer required continguous promoter and a fusion protein region of HDAC/GFP. Class discussion amplified on this.
T or F. All three methods of sequencing we have discussed depend upon synthesis of a complementary DNA strand.
T or F. All three methods of sequencing we have discussed depend upon the use of nucleotides that are modified to block further synthesis.
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.