Biotechnology Part 2: Homework
1. Answer the questions on the ppt.
2. List properties of DNA that make gel electrophoresis possible and meaningful.
a. Negatively charged- attracted to the positive end
b. Contain VNTRs or microsatellites that differentiate between individuals (RFLPS)
i. VNTRS are more important to DNA analysis because they are unique to each
individual. In most coding regions, analysis can only determine specific gene
mutations.
3. Why is staining with ethidium bromide for gel electrophoresis needed?
a. To visualize the DNA as it approaches the positive terminal
4. A fragment of DNA is composed of 100 000 bp. Assume that restriction endonucleases recognize
4-8bp sequences (Use 4). Calculate the expected frequency of cuts if the recognition site is 6 bp
long and the number of cuts.
a. 46 = 4X4X4X4X4X4 =4096
b. 100,000/4096= 24.1 (24 cuts)
5. The following fragments were obtained; 5.3kb, 2.1kb, 6.8kb,3.6kb, 1.1kb, 2.2kb. Order the
fragments from fastest moving to slowest moving.
Order fastest (smallest)- 1.1kb, 2.1kb, 2.2kb, 3.6kb, 5.3kb, 6.8kb
a. Which fragment will be found closest to the positive end
i. 1.1kb
b. Which 2 fragments might be difficult to isolate?
i. 2.1 and 2.2kb because they are so close in size
c. Sketch the gel
6. What would happen if the concentration of agarose is increased?
a. The DNA would migrate much more slowly. If too dense then the large fragments may
not migrate at all.
7. Why is taq polymerase used instead of DNA polymerase III in PCR?
a. It can survive the heating needed to pull apart the strands
8. What is done instead of using helicase in PCR?
a. Use heat
9. How can polymorphisms arise?
a. mutations
10. In RFLP analysis, why is it necessary to use a radioactive probe when running a gel as opposed to
not using a probe?
a.
In RFLP analysis, it is necessary to use radioactive probes as opposed to simply running a gel,
since RFLP is performed on the whole genome. A gel would not produce a distinguishable
pattern, since digestion and gel electrophoresis of the whole genome results in a smear on the
gel. Radioactive probes bind to specific complementary DNA sequences in a process known as
hybridization. This process will produce a pattern on the autoradiogram and will thus allow
distinguishable bands to appear, as opposed to a gel, which would provide a smear of DNA.
11. Why is RFLP not effective on degraded DNA?
a. Not effective because it requires the comparison of fragment lengths. Cannot be
properly assessed if DNA is not intact.
b. For example, if a fragment is cleaved in half to begin with and is then digested, it will yield
three smaller fragments rather than the two relatively larger ones that would be found had the
DNA fragment not initially been degraded. In addition, if the area to which a probe hybridizes
is degraded or missing, then that probe will not produce a band on the autoradiogram.
12. What is hybridization. How can it help to determine if a particular sequence is present in DNA?
a.
Hybridization: Complementary base pairing between strands of nucleic acids via hydrogen
bonding. Hybridization can tell whether a specific sequence is present in a DNA sample. The
probe can be engineered to anneal to the complementary DNA of specific sequence on the gel.
Since the probe is radioactive, it will appear on an autoradiogram—it has the ability to burn an
image on X-ray film. The detection of a specific sequence is accomplished in this manner.
13. Why would less semen be needed in a criminal investigation as compared to blood for DNA
fingerprinting?
a.
Less semen is required than blood for DNA fingerprinting because the density of the cells in
semen, and thus the amount of DNA present, is much greater than that found in blood.