Quizzes - PGM

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 5CAATTG3
  also recognizes 5GTTAAC3.
• 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 its 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 dont 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
Quiz 7 10 October 2007

• 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.

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
  thats 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 wont 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

1. When using a lambda vector, what step must occur
   after ligation and before introduction of DNA
   into the host bacterial cells? In vitro
   packaging
2. 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.
3. Name a method that is used to find the interest
   you want in a genomic library. Plaque or colony
   hybridization.
4. What feature of any good vector allows you to
   grow up enough of the clone you want? High copy
   number (or independent replication, although its
   really a combination of independent replication
   to high copy number).
5. 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

1. Name any one high capacity vector other than a
   cosmid. P1, PAC, BAC, YAC
2. 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.
3. What substrate used in chain termination
   sequencing causes the chain termination? The
   dideoxynucleotides
4. How are the four bases distinguished in current
   fluorescence-based sequencing techniques? Each
   base of the 4 ddNTPs is labeled with a different
   color.
5. 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

1. 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.
2. In EMSA, what 2 types of molecules interact to
   cause the mobility shift? DNA and protein.
3. What organism do you use to grow up enough
   reporter construct for a reporter assay in mouse
   cells? E. coli.
4. 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 2007 bonus points

1. Both ChIP and EMSA study the interactions of DNA
   with what type of molecule? Protein
2. Which of the two methods, ChIP or EMSA, traps
   interactions in vivo? ChIP
3. Which of the two methods uses a reversible
   covalent crosslink in the process? ChIP
4. Name two different ways in which the DNA of
   interest in the ChIP assay is identified? PCR
   and direct sequencing and microarray
   hybridization
5. 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

1. Name any two of the 3 features all good cloning
   plasmids have in common. Ori for independent
   replication, drug resistance, multiple cloning
   site, etc.
2. Which type of end prevents Exonuclease III
   digestion a) 5 overhang or b) 5 recessed?
3. 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.
4. What distinguishes any expression plasmid from
   other cloning plasmids? Includes a transcription
   cassette.
5. In what organism do you grow up enough of your
   cloning plasmid to work with? E. coli

21
Quiz 20 19 November 2007

1. 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).
2. Who are the other members of your powerpoint
   team?
3. Briefly list what you yourself (just you) have
   done so far to contribute to the power point
   assignment.
4. Do you think your team members would agree that
   your answer to number 3 is accurate?
5. 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 Mondays
  lecture.
• (There can be more than one answer. Use no more
  than 3 sentences.)