RI 2023 Prelim H2 Bio P3 QP

© RI 2023 Preliminary Examination 9744/03 [Turn over RAFFLES INSTITUTION 2023 Year 6 Preliminary Examination Higher 2 BIOLOGY 9744/03 Paper 3 Long Structured and Free-response Questions 14th Sept 2023 2 hours Candidates answer on the Question Paper. Additional Materials: Writing paper. READ THESE INSTRUCTIONS FIRST Write your index number, CT group & name in the spaces at the top of this page. Write in dark blue or black pen. You may use a HB pencil for any diagrams or graphs. Do not use staples, paper clips, glue or correction fluid. Section A Answer all questions in the spaces provided on the Question Paper. Section B Answer any one question in the writing paper provided. The use of an approved scientific calculator is expected, where appropriate. You may lose marks if you do not show your working or if you do not use appropriate units. At the end of the examination, hand in your essay question SEPARATELY. The number of marks is given in brackets [ ] at the end of each question or part question. This document consists of 13 printed pages. CIVICS GROUP CANDIDATE NAME INDEX NUMBER 3 S 0 3 2 Raffles Institution Internal Examination For Examiner’s Use Section A 1 / 28 2 / 12 3 / 10 Section B 4 or 5 / 25 Total / 75

2 © RI 2023 Preliminary Examination 9744/03 For Examiner’s Use Section A Answer all the questions in this section. 1 Immunofluorescence is a technique that scientists use to visualize the distribution and morphological appearance of specific proteins within cells. The indirect immunofluorescence technique involves: 1. The production of specific antibodies that bind to a specific antigen. These are known as primary antibodies. 2. The addition of the primary antibodies to a preparation of cells or tissues fixed on glass slides. 3. The addition of secondary antibodies labeled with fluorescence dye called FITC which will bind to the primary antibodies. 4. Visualization under a fluorescence microscope. Under the light of excitation wavelength, 450nm, FITC will fluoresce. Fig. 1.1 shows the principles of the indirect method of immunofluorescence. Fig. 1.1 Immunofluorescence was used to study cells undergoing cell division. Fig . 1.2 show s a micrograph of a cell. The lighter area shows the position of the fluorescence. Fig. 1.2

3 © RI 2023 Preliminary Examination 9744/03 [Turn over For Examiner’s Use (a) (i) Identify the cell structures that fluoresced in Fig . 1.2 and name the specific antigen (protein X) that the primary antibodies bind to. [2] cell structure specific antigen (protein X) (ii) Identify the stage of mitotic division that the cell in Fig. 1.2 is in and describe the events that occur at this stage. …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………[3] (iii) The secondary antibodies are goat antibodies that bind to the primary antibodies which are mouse antibodies. With reference to Fig. 1. 1, state the name of the specific site that the secondary goat antibodies bind to and explain why the secondary goat antibodies do not bind to other sites. …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………[3]

4 © RI 2023 Preliminary Examination 9744/03 For Examiner’s Use (b) To produce primary mouse antibodies which bind to protein X, the following steps are carried out. Step 1 produce protein X Step 2 inject protein X into a mouse Step 3 harvest plasma cells from the mouse Step 4 fuse plasma cells with tumour cells to produce an ‘immortal’ hybrid cell that produces antibodies Step 5 harvest specific antibodies Fig.1.3 illustrates how the primary mouse antibodies are produced. Fig. 1.3 (i) Explain why plasma cells alone cannot be used directly for the long term production of antibodies. ………………………………………….………………………………………………………… …………………………….……………………………………………………………………… ………………………………………………………………………………….………………… ………………………………………………………………………………….………………… ………………………………………………………………………………….………………… ………………………………………………………………………………………..………..[3] inject mouse with protein X ‘immortal’ hybrid cells

5 © RI 2023 Preliminary Examination 9744/03 [Turn over For Examiner’s Use (ii) The Hayflick limit is the number of times a normal somatic cell population will divide before cell division stops. In order f or the ‘immortal’ hybrid cells in Fig. 1.3 to divide indefinitely, the cells need to overcome the Hayflick limit. Explain how the fusion of the plasma cells with the tumour cells enable the ‘immortal’ hybrid cells to overcome the Hayflick limit. …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………[4] (c) In order for a mouse to produce primary mouse antibodies, protein X needs to be injected into it. Dr N, a relatively inexperienced scientist, was tasked to use genetic engineering to produce purified protein X. This was Dr N’s experimental approach. Step A harvest total chromosomal DNA from a human cell Step B design PCR primers to amplify the coding region of the protein X gene Step C perform PCR on the coding region of protein X Step D insert the coding region of protein X into a bacteria plasmid Step E insert the recombinant plasmid containing the coding region of protein X into Escherichia coli bacteria by a process called transformation Fig. 1.4 below summarises Dr N’s approach. Fig. 1.4

6 © RI 2023 Preliminary Examination 9744/03 For Examiner’s Use Dr N obtained the nucleotide sequence of the coding region of protein X gene from his supervisor, Prof S. The first and last 20 nucleotides of the template DNA strand are shown below. 3’ GTACAGTTAA CATTGTCACG ……………TGATAAAAGA GACATTTCAA 5’ (i) Using the information above, help Dr N to design a 10 nucleotide long ‘ forward’ and 10 nucleotide long ‘ reverse’ primer that can be used to amplify the coding region of the protein X gene. State the sequences of the respective primers in the table below. type of primer sequences of primer ‘forward’ ‘reverse’ [2] Dr N was tasked to set the PCR conditions to amplify the coding region of protein X. His proposal is shown below: stage temperature duration 1 65oC 30s 2 72oC 60s 3 95oC 90s Repeat stages 1, 2 and 3 sequentially for 30 cycles. (ii) Comment if Dr N’s proposed PCR conditions will successfully produce the desired PCR product. …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… …………………………………………………………………………………………………… ……………………………