2011 a Level H2 Biology P3 Ans

2011 A Level Paper 3 Suggested Answers PAPER 3 Question 1 (a) Isolate insulin mRNA from β-cells of islet of Langerhans in the pancreas; Using reverse transcriptase, synthesise a DNA strand using mRNA as the template;; Degrade the mRNA using RNAse; Using DNA polymerase, synthesise a second DNA strand using the first DNA strand as the template;; Forming a double-stranded cDNA; (b) Examiners’ Comments: Many candidates tried to use a cDNA library to obtain the required DNA. Since cDNA is only formed using mRNA, this approach is incorrect. Candidates who used the information in the question that somatostatin is a chain of 14 amino acids were able to give valid suggestions. Since somatostatin is a short polypeptide chain of 14 amino acids, its DNA can be chemically synthesised;; Nucleotides can be derived using the codon dictionary of the genetic code; and added one by one before ligation;; (c)(i) Cuts and destroy foreign DNA (e.g that of invading viruses) that enters the bacteria;; thus protecting the bacteria;; (ii) Sticky end 1: AATT Sticky end 2: CTAG (iii) To ensure the correction orientation of the gene (promoter upstream of stop triplet) when inserted into a vector;; (d) Incubate the plasmid with EcoRI and BamHI to allow restriction enzymes to cut at specific restriction sites, creating sticky ends complementary to that on the somatostatin gene;; Incubate somatostatin (with sticky ends added) with the cut plasmid to allow complementary ends to anneal via H bonding;; Add DNA ligase to catalyse the formation of phosphoester bonds between adjacent nucleotides, thus forming the recombinant plasmid;; (e) Small size facilitates the vector’s entry into host cells and the biochemical manipulation of DNA;; Presence of origin of replication allows for the vector to replicate itself and the inserted gene of interest within the host cell;;

2011 A Level Paper 3 Suggested Answers Question 2 (a)(i) Normal allele of Rpe65 gene can be targeted;; by the viral vector to pigment cells beneath the retina;; Normal allele of Rpe65 gene can be integrated into the host cell chromosome;; and be passed on to daughter cells during replication, offering an opportunity for long term stablilty of the allele;; Normal allele of Rpe65 gene can be introduced into the nucleus;; offering an opportunity of long-term stable expression of the allele;; (ii) Liposomes have no viral genes that may cause diseases in host cell;; Liposomes would not trigger attack by host immune system, thus normal allele of Rpe65 gene is not destroyed;; (b) Alleles delivered by adenoviruses are not integrated into chromosomes, thus can only function sporadically;; Alleles delivered by retroviral vectors integrate randomly into host cell chromosomes, so might disrupt useful genes in the host cell and cause disease;; This also means that therapeutic alleles would not be passed on the new cells, and would be lost as the cells die;; Reject: genes die or do not live for very long (c)(i) Examiners’ Comments: The question did not ask how a genomic DNA library was constructed or what it was used for. A random sample of all the DNA sequences in an organism;; Entire genome is cleaved with a specific restriction endonuclease;; Resulting fragments are inserted into plasmids before introducing into bacteria;; (ii) Examiners’ Comments: The question did not ask how a cDNA library was constructed or what it was used for. cDNA library contains only regions of the genome that have been transcribed into mRNA;; Total RNA from a specific tissue is converted, using reverse transcriptase, to a doublestranded cDNA;; These molecules are inserted into plasmid and cloned;; Question 3 (a) (i) Cells were grown in culture vessels containing nutrients and plant growth regulators;; Auxin/cytokinin is added to the nutrient agar to stimulate the cells to divide by mitosis forming a callus;; The number of calli increased by subculturing;; The cells of the callus are induced by auxin and cytokinin/gibberellin to differentiate into a plantlets;; producing large number of genetically identical plants

2011 A Level Paper 3 Suggested Answers (a) (ii) Examiners’ Comments: When beads of tungsten are fired at very high speed at corn cells, the cell wall, cell surface membrane or nuclear envelope will not be ‘too strong to prevent entry’. as the tungsten were fired at very high speed at corn cells, most of the cells will be damaged and only some cells will take up the plasmids;; The probability of the plasmid reaching the nuclei and be incorporated into the chromosome is low;; (b) (i) Growing a Bt crop could affect food chain. All of the Bt-susceptible caterpillars that fed on the Bt leaves died before the wasps could complete their development and emerged; as compared to 63% of Bt-susceptible caterpillars that fed on non-Bt leaves survived long enough for the adult wasps to emerge; (b) (ii) There is evidence that the Bt toxin kill wasp larvae; For Bt-resistant caterpillar that fed on Bt leaves, there are 54% of the parasitized caterpillars from which adult wasps emerged; which is 2% lower than for those fed on non-Bt leaves; Question 4 Planning Question An explanation of the theory to support your practical procedure [1] 1. The plasmid contains 2 selectable marker: ampicillin (AmpR) and tetracycline resistance (TetR) genes / Name the different types of cells present after transformation 2. There are multiple cloning sites in TetR gene. Restriction enzyme is used to cut at TetR gene to insert the gene of interest. 3. Insertion of gene of interest will inactivate the gene conferring resistance to tetracycline. 4. Transformed cells with recombinant plasmid will be AmpR but TetS 5. Transformed cells with non-recombinant plasmid will be AmpR but TetR. 6. Non-transformed cells will be AmpS. 7. Nucleic acid hybridisation to identify the cells with anti-thrombin gene inserted into the plasmid. Description of the method used including the scientific reasoning behind the method and the type of data generated by the experiment [9] (I) Identification of E. coli transformant cells that carry recombinant plasmids 8. Plating: Plate E. coli cells on agar plate containing ampicillin (master plate). a. to identify the transformed cells which have taken up plasmid from the nontransformed cells. Transformed cells will be AmpR 9. Replica-plating: use sterile velvet to replica-plate the cells obtained from above onto tetracycline-containing nutrient agar. a. to identify the transformant cells that have picked up the non-recombinant plasmids instead of the recombinant plasmids. b. the restriction fragments are inserted into the tetR gene in the plasmid. This will inactivate the gene conferring resistance to tetracycline/ insertional inactivation of an antibiotic resistance gene. Recombinant cells are AmpR but TetS. c. cells that carry non-recombinant DNA are resistant to both ampicillin and tetracycline.

2011 A Level Paper 3 Suggested Answers 10. Identification: Colony hybridisation to identify the transformed cells that carry the antithrombin gene. (II) Identification of E. coli transformant cells that carry the gene of interest by colony hybridisation. 11. Blotting: All the ampRtetS colonies collected from the ampicillin master plate are transferred onto the nitrocellulose filter and lysed to release DNA (using alkaline solution). a. the lysis treatment/alkaline solution results in the denaturation of DNA. b. makes it single-stranded for subsequent hybridization with probes 12. Probing: nitrocellulose membrane is exposed to radioactively-labelled, single-stranded DNA probes. a. radioactively-labelled, single-stranded DNA probes are designed to be complementary to the gene of interest, can hybridise via complementary base pairing. 13. Autoradiography: a sheet of photographic film is laid over the nitrocellulose membrane and developed after a suitable period of exposure. a. radioactive probes that are bound to the gene of interest form an image on the photographic film [total: 14, max 9] How results will be analysed including how the bacteria with recombinant plasmid can be determined [1] 14. Compare the master plate and replica plate, identify bacteria that are resistant to ampicillin but susceptible to tetracycline. These are the bacteria that have taken up recombinant plasmid. Colonies of recombinant bacteria can be isolated by going back to the original cultures on the ampicillin-containing agar. [½] 15. Compare the photographic film with the master plate. The colonies contain the gene of interest can be identified. [½] Scientific terms [1] Question 5 (a) 1. loading of DNA fragments at one end of the agarose gel; Ref to addition of loading dye to DNA fragments: 2. They increase the density of the sample, ensuring that the DNA sinks into the well; 3. They add colour to the sample to make it easy to load into the wells; 4. They contain tracking dyes that, in an electric field, move toward the anode just like the DNA fragments so it serve as visual markers for migration during electrophoresis and indicates when to turn off the power; 5. The gel is bathed in an buffer solution which contains ions that conduct electricity; 6. electric field or voltage applied across the gel; 7. DNA being negatively charged; 8. will move towards the positive electrode/anode; 9. rate of movement/speed/distance travelled in a given time depends on the size of DNA fragments; 10. larger DNA fragments travel slower and smaller fragments traveling faster; 11. more resistance for the larger fragment to move through the pores of the gel; 12. DNA ladder is added to calibrate; 13. use of ethidium bromide / fluorescent dyes and UV light to see the DNA bands;

2011 A Level Paper 3 Suggested Answers (b) Definition of RFLP 1. Variations in gDNA sequences among individuals of a species; 2. Caused by mutations found within coding or non-coding regions; 3. Variety of genotype / inherited genetic differences among individuals in a population/the varied alleles of the genes in the gene pool; 4. Giving rise to variations in length of DNA fragments when cut by the same restriction enzyme;; Detection of RFLP 5. Separation of restriction fragments of different sizes by agarose gel electrophoresis; Southern blot: 6. DNA fragments transferred onto nitrocellulose membrane; 7. Alkaline condition used to denature DNA, making it single-stranded; Probing: 8. Nitrocellulose membrane exposed to radioactively-labelled, single-stranded DNA probes; 9. Probes designed to be complementary to VNTRs; 10. Can hybridise / attach to restriction fragments of interest via complementary base-pairing; Autoradiography 11. Photographic film laid over nitrocellulose membrane and developed after suitable period of exposure; 12. Radioactive probes bound to restriction fragments form an image on photographic film;

(c) Examiners’ Comments: The majority of answers were in terms of the disease mentioned in the syllabus - sickle cell anaemia and these were often extremely detailed. Candidates who wish to describe other diseases should ensure that the complete details of restriction enzymes, fragment lengths etc. are known to the same degree as they are known for the well documented example of sickle cell anaemia.  -globin gene 1. Sickle-cell anaemia is caused by a base substitution within a restriction site in the human -globin gene;; 2. A thymine nucleotide was substituted by an adenine nucleotide; / triplet base code change from CTT to CAT;; RFLP analysis 3. A restriction enzyme recognises and cuts DNA molecules at a specific base sequence (i.e. restriction site;; 4. The point mutation destroys one of the DdeI restriction sites at the 5’ end of the -globin gene;; 5. When the normal and sickle-cell alleles of the -globin gene are cut with the same restriction enzyme DdeI;; 6. different mixture of fragments from each allele gives its own band pattern in gel electrophoresis;; 7. normal allele contains 2 restriction sites within the coding region, producing 3 bands;; 8. sickle-cell allele contains 1 restriction site within the coding region, producing 2 bands;; 9. At the end of nucleic acid hybridization, the band patterns of the person homozygous for normal alleles, homozygous for sickle-cell allele and heterozygote will be different;;

2011 A Level Paper 3 Suggested Answers 10. i.e. person homozygous for normal allele, produces 3 bands; person homozygous for sickle-cell allele, produces 2 bands; heterozygote, produces 4 bands;

Alternative restriction enzyme, e.g. MstII (For details pls ref to N08/P3/Q1)