What you will be expected to know:
1. Know that a restriction enzyme cuts specific sequences of DNA – I will stick to 6-bp
sequences that are palindromes. Be able to do the type of question given for lab #7 – the
restriction enzyme handout.
2. Understand how creating recombinant DNA happens – see Figure 20.43 in the text
(which was in the lecture) and the one I had in the lecture about starting with RNA and
using reverse transcriptase. So I would give you the plasmid with restriction sites, the
starting material (RNA or DNA), etc. and you would need to tell me how to get the DNA
into the plasmid and how you would pick bacteria clones that are likely to have your
recombinant plasmid.
3. Know how PCR works – what is put into the tube, and how the DNA is amplified.
What is special about the polymerase used? If you started out with 1 DNA molecules,
how many are theoretically present at the end of 10 cycles?
4. Uses for PCR – DNA fingerprinting. How do you use PCR to identify a person?
5. Gel electrophoresis – why does DNA migrate? In which direction does it migrate? Be
able to “read” a gel – identify sizes of bands and make conclusions.
6. Figure 20.9 is a great example of something I would ask – give you the info about the
mutation (eliminating a restriction enzyme site) – then give you the gel and ask you to
determine someone’s genotype.
7. This continues on to Southern blotting – how does this technique work? How would
you set it up? What is the purpose? See example of how it is used in DNA fingerprinting.
8. Understand how dideoxy sequencing is done – the use of dideoxynucleotides to create
fragments of DNA of different lengths. How would you set up the reactions to sequence a
fragment of DNA?
9. Gene chips – what is the technique – i.e. steps involved – and what information do you
obtain from this technique?
Questions:
1. Specific DNA sequences or bands are identified in a Southern blot by hybridization
with
a. a vector
b. an antibody
c. a virus
d. a probe
2. The first step in the polymerase chain reaction is
a. denaturation
b. primer extension
c. annealing
d. cooling
3. Viruses containing RNA and reverse transcriptase are called
a. riboviruses
b. bacteriophages
c. rotaviruses
d. retroviruses
4. When sticky ends are paired, they can be joined by
a. restriction enzymes
b. methylase
c. DNA ligase
d. polymerase
5. In the initial screening of bacterial clones, a clone that has metabolized or cleaved Xgal is
a. yellow
b. orange
c. blue
d. green
6. What does it mean if the bacterial clone is white? What does it mean if it is a different
colour?
7. DNA molecules can be cut into fragments by:
a. restriction enzymes
b. ATP
c. plasmids
d. Taq polymerase
8. PCR is used to
a. cleave plasmids
b. amplify small amounts of DNA
c. seal “sticky ends”
d. none of the above
9. Which of the following statements about cDNA is true?
a. mature mRNA directs the formation of cDNA
b. mature mRNA does not contain introns
c. DNA taken from the nucleus is used to produce cDNA
d. a and b
e. none of the above
10. What enzyme is used to make cDNA from an mRNA template?
11. Genetically identical organisms derived from a single genetic source are called
a. populations
b. varieties
c. clones
d. subtypes
12. The autoradiograph from a Southen blot (below) shows the results from a single locus
probe DNA fingerprint analysis for a man and woman and their four children. Which
child is least likely to be the biological offspring of this couple? Why?
13. The autoradiograph from a Southen blot (below) shows the results from a single locus
probe DNA fingerprint analysis for a man and his four children.
Which lane contains the DNA of the father?
Explain why.
14. Explain the technique used to obtain the autoradiographs in questions 12 and 13. You
are given genomic DNA of each individual and a specific restriction enzyme. Explain the
rest in detail.
15. A eukaryotic gene (MEK1) has "sticky ends" produced by the restriction
endonuclease EcoRI. A plasmid, which contains genes for resistance to ampicillin and
tetracycline (antibiotics), has one recognition site for EcoRI located in the tetracycline
resistance gene. The plasmid is cut with EcoRI and a mixture of cut plasmid and the
MEK1 with “sticky ends” is incubated for several hours with DNA ligase. This mixture is
then used to transform bacteria, which are grown on nutrient broth (LB). The bacteria are
allowed to grow overnight and are streaked on a plate using a technique that produces
isolated colonies that are clones of the original. Samples of these colonies are then grown
in four different media: LB + ampicillin, LB + tetracycline, LB + ampicillin and
tetracycline, and LB without antibiotics.
A. If the bacteria are transformed with plasmid that has not incorporated MEK1, in what
media would they grow in?
B. What media would bacteria containing a plasmid into which the eukaryotic gene has
integrated would grow?
C. If the bacteria have not been transformed with plasmid, in which media would they
grow?
D. If the MEK1 to be inserted into the plasmid is the coding sequence with no introns,
suggest one way that it may have been isolated.
16. Describe in detail how you would sequence the MEK1 insert in the plasmid – assume
you have a primer that is specifically recognizes a sequence of DNA in the plasmid that is
upstream (5’ to) where the MEK1 has been inserted.