2016 VJC H2 Biology Prelims Paper 3 Answers

1 Question 1 (a) The bacterial plasmid, pBR322, was used as a vector for Gene X as shown in Fig. 1.1 below. Gene X was inserted in the BamHI restriction site. pBR322 also contain the EcoRI and HpaI restriction sites. The target sites for these restriction enzymes are shown in the table below. The lines drawn in each sequence show where the enzyme cuts the DNA molecule.     (i) With reference to Fig. 1.1, explain how two properties of plasmid pBR322 allow it to be used as a vector. [2]  Presence of origin of replicati on; so inserted gene can be replicated;  Has ampicillin and tetracycline resistanc e genes; as selection markers / allow identification of host cells that have successfully taken up the recombinant plasmid (ii) Outline the steps taken to produce the recombinant plasmid shown in Fig. 1.1. [2]  Cut plasmid and gene X with BamHI restriction enzyme;  “Sticky ends” generated;  Allow plasmid and gene to anneal wit h ref. complementary base pairing; 2016 H2 Biology Prelim Paper 3  ANSWERS

2  Add ligase to seal the nicks in the sugar-phosphate backbone / form phosphodiester bonds between the ends of the cut plasmid and gene; (iii) Explain the disadvantage that would arise if gene X was to be inserted into the HpaI restriction site instead of the BamHI site. [2]  HpaI generates “blunt ends”;  No hydrogen bonds to hold t he cut plasmid and gene together;  Ref. extra step that will require linker;  To generate “sticky ends” (b) Calcium chloride heat shock treatment wa s then used to introduce the recombinant plasmid into Escherichia coli . However, the process of creating recombinant plasmids is typically not 100% efficient. Often, a mixture of re-annealed plasmid and re-annealed DNA is produced along with the recombinant plasmid. These may be taken up by the bacteria as well. This nece ssitates the process of selecting for the bacteria that have successfully taken up t he recombinant plasmid. As such, the bacteria was first plated onto a nutrient agar plate containing ampicillin. Replica plating was subsequently carried out onto a nutrient agar plate containing tetracycline. Bacterial growth on both plates is shown in Fig. 1.2. With reference to Fig 1.2, (i) Circle the colonies that were successf ully transformed with the recombinant plasmid. [1] (ii) Account for the difference in colony numbers in Plate A and B. [3]  Plate A selects for all successfully transformed cells/ taken up plasmid with ampicillin gene; (maybe recombinant plasmid or re-annealed plasmid);  Selected against / killed off cells which took up re-annealed DNA;  Plate B contain 3 fewer col onies compared to Plate A;  Missing colonies had successfully ta ken up the recombinant plasmid;  where gene X had been inserted into tetrac ycline gene and disrupted it (ref. to insertional inactivation) / colonies lost tetracycline resistance and died;

3  Therefore comparing the difference in colony numbers between Plate A and B will allow us to identify recombinant cells/ colonies; (c) In a separate cloning experiment, E.coli cells were transformed with another type of plasmid carrying a different selectable marker . Fig. 1.3 shows the results of plating the transformed E.coli cells onto an agar plate with the appropriate substances. Fig. 1.3 (i) Explain why some colonies appeared white while others appeared blue. [4]  The cells must have been transformed by plasmids containing intact lacZ gene as the selectable marker;  The agar plate must have contained X-gal and IPTG;  lacZ gene codes for β-galactosidase enzyme;  β-galactosidase enzyme catalyses the conversion of X-gal in the agar from colourless to blue;  Hence, colonies that contain the reannea led / non-recombinant plasmids appeared blue.  Should the lacZ gene in the plasmid be disr upted as a result of insertion of foreign DNA / gene, ref. to insertional inactivation of lacZ gene;  no functional β-galactosidase enzyme will be produced and thus no blue product is formed;  Hence, colonies that contain the recombinant plasmids appeared white.

4 (ii) Suggest why replica plating was not necessary in this experiment. [1]  By their colours, colonies that contai n the recombinant plasmids (appeared white) can be differentiated from those that do not (appeared blue);  Replica plating is necessary only when t he selection process kills off the desired colonies.  In the case of using two antibiotic resi stance genes as the selection markers, the insertion of foreign DNA / gene disrupts only one of the two antibiotic resistance genes in the plasmid.  To identify colonies that contain the re combinant plasmids, the colonies must be treated with two types of antibiotics.  Those that survived one ant ibiotic treatment but not the other would be the desired colonies.  However, adding both antibiotics to the same agar plate would kill off the desired colonies.  To get living cells that contain the reco mbinant plasmids, replica plating must be done. ) d) In a separate cloning experiment, another plasmid pBR33 was used to introduce Gene Z into a different strain of E.coli bacteria. Gene Z was inserted into one of the three genetic markers found in pBR33 – neomycin resistance gene, kanamycin resistance gene and streptomycin resistance gene. The bacteria were then plated onto nutrient agar plate containing neomycin. Replica plating was subsequently carried out onto nutr ient agar plate cont aining streptomycin. Bacterial growth on the two plates is shown in Fig. 1.4 below. Account for the results obtained in Fig. 1.4. [3]  Gene Z inserted in kanamycin-resistant gene;  Insertional inactivation;  3 functional genes in re-annealed/non-reco mbinant plasmids vs 2 functional genes is recombinant; Replica plate containing Streptomycin (Plate S) Plate containing Neomycin (Plate N)

5  Bacteria with recombinant plasmid can survive in presence of neomycin and streptomycin  Bacteria with re-annealed plasmid can survive in all 3 antibiotics  Both plates have same number; and posit ion; of colonies; QV: 10 colonies  Both plates do not contain any non-trans formed bacteria; consists of transformed cells with recombinant and non-recombinant plasmid Alternative:  Gene Z inserted neomycin-resistant gene;  So plate N has killed off all bacte ria with recombinant plasmids and non- transformed bacteria  So when is done on from Plate N to plate S, only the bacteria that survive (i.e. the recombinant bacteria) are picked up by the nitrocellulose membrane and transported to plate S; [Total: 18] Question 2 Explain why two primers are used for polymerase chain reaction. [2]  Ref. to the 2 primers as forward and reverse primers [1/2]  Flank the targeted sequence to be amplified [1/2]  Amplify large quantities of a specific sequence of DNA in a short period of time [1/2]  1 primer anneals to 1 of the separated DNA strands after denaturation [1/2]  Provide free 3’ OH for Taq polymerase to elongate the complementary strands of both templates to produce 2 DNA molecules [1/2] Duchenne muscular dystrophy is a genetic disease in which t here is a progressive loss of muscle mass, leading to physical weakness, difficulty in standing and walking, and eventually paralysis and death. Early symptoms of the di sease can only be observed between the ages of 2 and 3 in most patients. A group of doctors and medical biologists discovered a RFLP marker, found on the sa me chromosome as the disease gene, which can be used in the screening of the disease during pregnancy. To investigate the effective of the RFLP marker in disease screening, samples of DNA were obtained from a family known to have t he disease. The RFLP locus was isolated and amplified using polymerase chain reaction,