2015 RI H2 Biology Prelims Paper 3 Answers

© RI 2015 Preliminary Examination 9648/03 [Turn over RAFFLES INSTITUTION 2015 Year 6 Preliminary Examination Higher 2 BIOLOGY 9648/03 Paper 3 22nd SEPTEMBER 2015 2 hours Additional materials: Answer Sheet READ THESE INSTRUCTIONS FIRST Write your index number, CT group & name on all the work you hand in. Write in dark blue or black pen on both sides of the paper. You may use a soft pencil for any diagrams, graphs or rough working. Do not use staples, paper clips, highlighters, glue or correction fluid. Sections A and B Answer all questions. At the end of the examination, hand in your essay 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 5 S 0 3 1 For Examiner’s Use Section A 1 /12 2 /13 3 /15 4 /12 Section B 5 /20 Total /72 Raffles Institution Internal Examination

2 © RI 2015 Preliminary Examination 9648/03 For Examiner’s Use Section A Answer all the questions in this section. 1 Plasmid X is used to clone a gene of interest. Fig. 1.1 shows a diagram of plasmid X. The restriction sites of four restriction enzymes are shown in Table 1.1. Fig. 1.1 Restriction Enzyme Restriction Sites AaaI 5' ---T GTACA--- 3' 3' ---ACATG T--- 5' Acc65I 5' ---G GTACC--- 3' 3' ---CCATG G--- 5' HaeIII 5' ---GG CC--- 3' 3' ---CC GG--- 5' XmaI 5' ---C CCGGG--- 3' 3' ---GGGCC C--- 5' Table 1.1 (a) (i) With reference to the information in Table 1.1 , describe the characteristics of a restriction site. [2] 1. Restriction site is a specific DNA sequence consists of 4 to 6 base pairs ; 2. which is palindromic* where reading from 5’ to 3’ (or 3’ to 5’) of each of the strands yield the same sequence

3 © RI 2015 Preliminary Examination 9648/03 [Turn over For Examiner’s Use (ii) The restriction enzyme PuvII recognises a 6 base pair sequence o n double stranded DNA. The first three bases of one strand are given, complete the restriction site for PuvII. [1] 5’ C A G _ _ _ 3’ 3’ _ _ _ _ _ _ 5’ (iii) Discuss the significance of having the multiple cloning site on plasmid X. [1] 1. Presence of MCS that has a number of UNIQUE different restriction sites on a plasmid allows the plasmid to be used to insert / contain a wide range of different foreign genes; (b) To clone a gene of interest into a plasmid, a researcher used restriction enzyme AaaI to digest the plasmid. However, he used restriction enzyme Acc65I to isolate the DNA fragment. (i) Explain whether or not the DNA fragment can be cloned into the plasmid to form a recombinant plasmid. [3] 1. Yes the cut DNA by Acc65I can join to plasmid cut by AaaI 2. as they both produce complementary / same sticky ends with GTAC / CATG 3. that can form hydrogen bonds and anneal* by complementary base pairing* 4. Adenine pair with thymine and cytosine with guanine (ii) In another experiment, HaeIII was chosen over the other 3 restriction enzymes to produce restriction fragments. Explain an advantage of using HaeIII in cloning. [2] 1. HaeIII produces blunt ends* 2. Plasmid / Restriction fragments produced will not reanneal*. 3. Less specific , can ligate into any blunt -ended DNA fragments of interest (ref to wider range of plasmid / fragments to clone) 4. Ref to 4base cutter vs 6 base cutter – shorter fragments, easier to clone Must have 1 (c) Plasmid Y was digested with EcoRI, BamHI, and both restriction enzymes together. All reactions are allowed to run to completion and Table 1.2 shows the sizes of the resulting DNA fragments: EcoRI BamHI EcoRI and BamHI 7.0 kb 4.0 kb 4.0 kb 1.0 kb 2.8 kb 2.0 kb 1.2 kb 1.0 kb 0.8 kb 0.2 kb Table 1.2 (i) Using the circle below, draw a restriction map for plasmid Y. [2]

4 © RI 2015 Preliminary Examination 9648/03 For Examiner’s Use Can be clockwise / anti clockwise (ii) During the digestion of the DNA by HaeIII, regulation of temperature is important. An experiment was carried out to investigate the rate of digestion by restriction enzyme , HaeIII, over a range of temperatures. Explain the significance of carrying out this experiment. [1] Identify optimum temp, where activity is the highest ; so as to achieve complete digestion at the fastest rate / shorter time;. . [Total : 12]

5 © RI 2015 Preliminary Examination 9648/03 [Turn over For Examiner’s Use 2 Restriction fragment length polymorphism ( RFLP) can be used to detect variations in genomes. The technique is widely used in molecular biology. (a) Explain what RFLPs is. [3] 1. Due to the presence of DNA polymorphisms/nucleotide differences in homologous regions/loci/marker of different individuals 2. Resulting in variations in number and/or location of restriction sites and 3. Varying number of tandemly repeated nucleotide sequences among individuals 4. when digested by restriction enzymes* , produce fragments of different length and/or number / unique banding pattern among individuals. (b) Women with mutations in the BRCA–1 gene found on chromosome 17, have been found to have a significantly increased risk for breast and ovarian cancers. These mutations are often heritable. Women with family members who show incidences of breast and ovarian cancers sometimes choose to get tested to determine if they have a mutated BRCA-1 gene. Genetic testing typically involves RFLP analysis. A radioactive probe, P, revealed two RFLP alleles that are tightly linked to the BRCA-1 gene. Both RFLP alleles are shown in Fig. 2.1 . The arrows represent the restriction sites of the restriction enzyme used. The size of each resulting fragment (in kb) is indicated. Fig. 2.2 shows the pedigree of a family with some female members with incidences of breast and/or ovarian cancer, as well as the results of their RFLP analysis. The pedigree and RFLP analysis was prepared at the request of individual III-1 who requested medical adv ice about her risk for developing cancer. Each person’s DNA sample is shown directly below the individual. Fig. 2.1

6 © RI 2015 Preliminary Examination 9648/03 For Examiner’s Use The underlined numbers indicate the age at which cancer developed in individuals I-2, I-5, II- 3, II-4, III-2 and III-5, where Br indicates incidence of breast cancer and Ov for incidence of ovarian cancer. The current ages of II-2 and III-1 have also been indicated. Fig. 2.2 (i) Based on the Southern blot results shown in Fig. 2.2, indicate with a box