Cavendish campus

SCHOOL OF BIOSCIENCES
EXAMINATION MARK SCHEME
Module Code:
Module Name: Gene Manipulation
Date:
May/June 2004
With all questions some marks (approx. 20%) for each question are given for communication skills(planning, correct punctuation, good spelling, English usage and standard of scientific English, correctpresentation of Latin names); originality and creativity in the development of the answer. Unless specified,the student must discuss rather than describe. Points given for essay questions should generally beprefixed by the wording “The student should ..”.
1. (Compulsory
question)
Briefly explain the results for all 4 procedure used in the experiment as shown in Table 2.
Procedure 1: Explain that host cells cannot grow on tetracycline agar because the repressor geneis intact and its product inactivates the tetracycline gene. Host cells cannot grow on ampicillin agarbecause this vector lacks an ampicillin resistance gene.
Procedure 2: Explain that the insert disrupts the repressor gene by insertional inactivation allowingthe tetracycline gene to be expressed and making the host cells tetracycline resistant and ableto grow on tetracycline agar. The plasmid has no ampicillin resistance gene and, therefore, thehost cells are ampicillin sensitive and cannot grow on ampicillin agar.
Procedure 3: Explain that the insert disrupts the O/P site of the tetracycline gene preventing thebinding of RNA polymerase and making the host cel s tetracycline sensitive and unable to growon tetracycline agar. The plasmid has no ampicillin resistance gene and, therefore, the host cellsare ampicillin sensitive and cannot grow on ampicillin agar.
Procedure 4: Explain that the insert disrupts the tetracycline gene by insertional inactivationmaking the host cells tetracycline sensitive and unable to grow on tetracycline agar. The plasmidhas no ampicillin resistance gene and, therefore, the host cells are ampicillin sensitive and cannotgrow on ampicillin agar.
Of the 3 unique restriction sites of the plasmid used in the experiment, which is the best
one to use for gene manipulation work, and why?
The HaeIII site in the repressor gene. Inserts at this site allow recombinants to be selected
positively on tetracycline agar without the need for replica plating.
Explaining your reasoning, suggest two restriction enzymes, other than BamHI, that could
be used to produce DNA fragments with ends compatible for insertion into the tetracycline
gene of this plasmid.
BamHI gives GATC 5' overhangs. Therefore, any other restriction enzyme that produces the same
type of overhang could be used. There are 3 possible enzymes given in Table 1, viz, BalII, BgllI,
MboI.
If gene X had EcoRI ends, briefly explain how it could be inserted into the repressor gene
of this plasmid.
By converting the EcoRI sticky ends into blunt ends using either an exonuclease or DNA
synthesizing enzyme. The use of linkers is inappropriate here.
What are the main advantages of the use of a plasmid of this type for gene manipulation
compared to pBR322?
It allows positive selection on tetracycline agar without the need for replica plating thus saving
time and work. pBR322 is a negative-selection vector and requires replica plating and two
incubations. Student may also mention that the HaeIII site allows any blunt-ended fragments to
be inserted at this site (but at low efficiency) without the need to consider the complementarity
of sticky ends.
If gene X was the mean size of insert that this plasmid can accommodate, what would be
the actual number of clones required to construct a gene library for the colonial protozoan,
Dictyostelium discoideum, which has a genome 34 million bases in size with a 95%
probability of including any given sequence?
Minimum of clones required (n) = genome size (kb) SCHOOL OF BIOSCIENCES
EXAMINATION MARK SCHEME
Module Code:
Module Name: Gene Manipulation
Date:
May/June 2004
Actual number of clones (N) = ln (1-P) = = -2.9957323 = 1.20 x 104
What would be the actual number of clones required if a bacterial artificial chromosome
vector (mean insert size = 300 kilobases) was used instead of this plasmid for a
Dictyostelium discoideum gene library with the same probability (95%) of including any
given sequence?
-2.9957323 = 338.01
Discuss the principles and the applications of the detection of either:
restriction fragment length polymorphisms;
or
nucleic acid sequences unique to a microbial species.
Outline how polymorphisms can be detected using restriction enzymes, blots and probes.
Use named examples to discuss the use of RFLPs to diagnose disease, e.g. CF, sickle cellanaemia, muscular dystrophies.
Explain what is meant by a ‘unique’ sequence and how this can aid species identification.
Provide examples of sequences that can be use to identify microorganisms, e.g. 16S, IS.
Outline methods for the detection of unique sequences, e.g. PCR, LCR, probes.
Use named examples to discuss the identification of microorganisms by targeting a unique NAsequence, e.g. Mycobacterium tuberculosis, Chlamydia trachomatis, Campylobacter, HIV.
Using named examples and providing possible solutions, discuss the potential problems
of producing eukaryotic proteins using prokaryotic host cells.
(Avoid any discussion of technician error and contamination.)
Cover a good selection of potential problems, e.g. wrong insert size, wrong vector, small peptidedegradation, reading frame out, no post-translational modifications, intron problems, wrongpromoter, codon differences, toxic products, no product export.
Give solutions to problems in all cases, e.g. vector change, host cell change, use of fusedproducts, reading frame corrections, intron excision, mature mRNA use, change promoter, codondifference correction.
Discuss the use of eukaryotic host-vector systems, e.g. YAC and yeast.
Discuss the physico-chemical conditions that affect the specificity of binding between two
single-stranded molecules of nucleic acid and the implications of this to the use of probes
and primers.
Explain the terms ‘specificity’ and ‘binding’ and introduce ‘hybridization’, ‘complementarity’ and‘mismatch’.
Define ‘stringency’ and cover the conditions that affect it, viz. pH, temperature, salt concentration,presence of denaturants.
Preferably provide a graph of % hybridization against, e.g. temperature.
SCHOOL OF BIOSCIENCES
EXAMINATION MARK SCHEME
Module Code:
Module Name: Gene Manipulation
Date:
May/June 2004
Discuss the significance of the mid-point temperature.
Define the terms ‘probe’ and ‘primer’.
Provide a range of examples of methods that involve NA hybridization, e.g. probes in blots andin situ; primers for various techniques including: PCR, site-directed mutagenesis, and synthesisof complementary DNA.
Explain that changing stringency can alter the binding of probes and primers to their target and,hence, the specificity of the method and its applications.
Critically discuss the following statement:
“The British government is wrong to allow the trial of GM maize because recombinant DNA
technology is dangerous and has produced no benefits for mankind.”
Define ‘recombinant DNA technology’ and GM (Gene Manipulation).
Provide a brief history/background of rDNA technology, including the recent announcement of GMcrop trials, perhaps mentioning the “landmark” discoveries and their dates.
Outline the main techniques used in rDNA and GM such as SDM, host-vector systems,sequencing, PCR and probes.
Use examples to discuss the undoubted benefits of GM technology now and in the future onmedicine (both diagnosis and treatment of inherited, infectious disease and cancer), agriculture,industry, biotechnology (transgenic organisms for the production of pharmaceutical products e.g.
insulin, growth hormone, vaccines, interferons), forensic science, transplantation, environmentalpollution and waste management.
Discuss safety issues associated with GM, how work can be made safer, e.g. biologicalcontainment, disabled host strains, and the safety record so far.
Discuss ethical issues (e.g. corporate control, life insurance, patenting of genes) and publicconcerns.
Provide a balanced discussion and definite conclusions to the statement.
Keith Redway
March 2004

Source: http://users.wmin.ac.uk/~redwayk/lectures/documents/04-MAYMS.pdf