RI Nov 15 H2P3 ans

Raffles Institution Nov 2015 (H2 Biology) Paper 3 (for 9744 syllabus) 2017 Nov 2015 H2 Biol Paper 3 N15P3Q1 The polymerase chain reaction (PCR) is a three-stage process.Describe what occurs in (a) (i) The first stage at 95°C, [2] 1. heating to 95C separates two strands of the DNA double helix; 2. by breaking the hydrogen bond between complementary bases through increased molecular vibrations, denaturing the DNA; (ii) the second stage. [2] 1. Cool to around 64C, for DNA primers to anneal; 2. Primers anneal to complementary 3’ end of each template/single strand; (b) (i) Suggest and explain why Taq DNA polymerase is now obtained from genetically modified E.coli. [2] 1. The use of genetically modified E.coli would allow for mass production of Taq DNA polymerase; 2. Taq polymerase can be easily obtained; (ii) With reference to your knowledge of PCR, explain why a half -life of 40 minutes at 95°C allows many cycles of PCR before the enzyme needs to be replaced. [3] A single cycle of PCR is very short/much shorter than 40 minutes; 1. Many cycles of PCR can occur within a single half-life of Taq polymerase; 2. As sufficient amounts of enzyme are present to carry out many cycles of PCR before replacement is required; (c) Explain how RFLP analysis of DNA samples from imported grain and non-GM grain can show whether or not the imported grain has been genetically modified. [6] 1. Cut the DNA samples from the imported grain and non -GM grain with same restriction enzyme* to obtain different-sized DNA fragments; 2. DNA is separated according to size in gel electrophoresis where negatively- charged DNA* migrates towards the positive electrode/anode when subjected to an electric field / current; 3. Meshwork of agarose fibres impedes movement of longer fragments more than shorter fragments resulting in smallest fragments moving f urthest/largest fragments least far from well; 4. Carry out Southern blotting using radioactive probe* for VNTR/STR repeat sequence followed by visualising the banding pattern using autoradiography / x- ray film 5. Banding pattern in DNA fingerprint is due to different alleles/markers producing different bands in gel resulting in the unique banding pattern for different DNA samples; 6. Different bands arise due to polymorphic nature of different DNA samples, there will be variations in number and location of restr iction sites and number of tandemly repeated nucleotide sequence among different DNA samples; 7. Genetic fingerprint of imported grain can be compared against fingerprint of non - GM grain to see how closely related they are; 8. If fingerprint pattern is similar between the two different DNA samples, then imported grain is non-GM; [Total: 15 ]