2016 FRQ by topics Core topics DNA and Genomics ANS Ms Toh

DNA and Genomics 1. 04/2/7 Outline the differences in structure between amino acids and nucleotides. [6] 2. [11/2/9c] Outline the main features of DNA replication. [8] (refer to ans in next qns) Describe the process of DNA replication and the experimental evidence for semi - conservative replication. [15] (b) 1. A portion of double helix is unwound and unzipped at the origin of replication by DNA helicase and hydrogen bonds between parental strands are broken. 2. Each strand is bound and stabilised by single-stranded binding proteins , preventing them from rewinding behind the replication fork. 3. DNA topoisomerase introduces a break in a single strand, thus allowing the strand to rotate around the break , and reseals the strand , thus eliminating positive supercoil in front of the replication fork. 4. Each parental strand acts as a template for the synthesis of new daughter strand. 5. Primase synthesises RNA primers in the 5’ to 3’ direction by adding ribonucleotides via complementary base pairing using parental DNA strand as template. 6. DNA polymerase adds deoxyribonucleotides to 3’OH end of RNA primer, via complementary base-pairing with the parental strand and catalysing the formation of phosphodiester bonds between nucleotides. 7. Adenine (A) always pairs with thymine (T) with two hydrogen bonds and guanine (G) always pairs cytosine (C) with three hydrogen bonds.

8. The leading strand is synthesised continuously in the 5’ to 3’ direction and the lagging strand is synthesised discontinuously, via a series of Okazaki fragments in the 5’ to 3’ direction. 9. The RNA primers are excised and replaced with deoxyribonucleotides by another DNA polymerase. 10. DNA ligase catalyses the formation of phosphodiester bond between the two Okazaki fragments. 11. The product of semi-conservative replication is two DNA daughter molecules formed from one original parental DNA molecule and each daughter molecule contains one strand conserved from the parental molecule and one newly synthesised strand.

Experimental evidence for semi-conservative replication 1. Grow Escherichia coli in a medium containing ammonium chloride (NH4Cl) with a heavy nitrogen isotope / 15N isotope, for many generations; 2. Every time a cell divides, its DNA replicates and 15N is incorporated into nucleotides which are used to synthesize new DNA. All the bases in the DNA molecules contain 15N and their DNA will be ‘heavy’. 3. Bacteria were transferred into a medium containing only the lighter nitrogen isotope / 14N isotope and allowed to grow. At various times of one, two or more generations after the transfer, samples of bacteria were collected; 4. DNA samples are extracted from bacteria of each generation and put into a caesium chloride solution and spun at 40000 g in a centrifuge; 5. The caesium chloride molecules sink to the bottom of the test tubes creating a density gradient. The DNA molecules will position at their corresponding level of density where its density equals to caesium chloride solution; 6. DNA molecules containing 15N are heavier than those containing 14N, so they ended up nearer the base of the tubes. 7. The tubes are observed under UV rays. DNA appear as fine layers in the test tubes at different heights according to their density; 8. By semi-conservative replication, DNA of first generation would be of intermediate density as all DNA molecules comprise one 15N strand and one 14N strand. 9. Half of DNA molecules from second generation would be of intermediate density and half of DNA molecules would be of light density as 50% of DNA molecules comprise one 15N strand and one 14N strand and 50% of DNA molecules comprise two 14N strands. 10. Annotated diagram.

Results proving DNA replication is semi-conservative Marker’s comments: Many students misinterpreted the question and went out to describe the process of semi - conservative replication. For those students w ho managed to elaborate on the experimental evidence, many are confused about the sequence of the usage of 14N and 15N in the Meselson and Stahl experiment. Many had the misconception that DNA was extracted from the cell and grown in the 14N / 15N medium. Most students were not able to describe the centrifugation progress and identified the generations incorrectly.

3. Predict and explain the results of the 3 models of DNA repl ication up to 2 generations.[10] In semi-conservative replication model, original parental molecule consisting of both N15 strands separate, both then serve as template for G1; In the first generation / G1, all DNA molecules consist of one original parental N15 strand and a newly synthesized N14 strands, which will be seen as one N14N15 band of intermediate density; In the second generation / G2, both strands in G1 molecule separate, each strand of N14 and N15 then serves as template for synthesis of new complementary strand such that 50% G2 molecules consist of N14N14 (one N14N14 band of light density) and 50% consist of N14N15 (one N14N15 band of intermediate density); In conservative model, in G1 there will be a N14N14 band and a N15N15 band of equal thickness; This is because the parental molecule remains intact and the resulting daughter molecule is formed from two newly synthesized DNA strands from N14; In G2, there will still be a N15N15 band as the two parental strands reassociate to restore the parental molecule, but a thicker N14N14 band as both strands of the DNA molecule act as templates for the synthesis of an entirely new DNA molecule using N14 so there will still be more DNA molecules made up of only N14; In dispersive model, in G1 there will be only one N14N15 band;

This is because the parental DNA molecule breaks up into short segments, which act as templates for the synthesis of DNA. The segments are then joined together, resulting in both old and new DNA interspersed along each strand in both daughter DNA molecules. Hence each strand of both daughter molecules contains a mixture of old and newly synthesised DNA; In G2, there will still be only one band but due to more incorporation of lighter N14, the single band will still be lighter than a N14N15 band but heavier than a N14N14 band; 4. Describe the structure and roles of DNA and RNA (tRNA, rRNA and mRNA). 1. DNA is made up of (deoxyribo) nucleotides, which comprise of a phosphate group, nitrogenous bases and deoxyribose as its pentose sugar; 2. DNA molecule consists of 2 chains / strands / polynucleotides that are anti-parallel and spiral around an imaginary axis to form a double helix; NOT wound around each other! 3. It has a uniform width of 2 nm with nitrogenous bases stacked 0.34 nm apart and there are 10 base pairs in each turn of the helix; 4. Hydrophobic nitrogenous bases face the interior while the hydrophilic sugar - phosphate backbones face the exterior / nucleotides linked by phosphodiester bonds to form sugar-phosphate backbone; 5. 2 chains / strands held together by hydrogen bonds which ar e formed via complementary base-paring, Adenine = Thymine and Guanine ≡ Cytosine; 6. Each DNA molecule carries genes and each gene is a unit of inheritance / stores coded instructions for the synthesis of RNA or protein / carries genetic information that is inherited; 7. RNA is made up of (ribo) nucleotides, which comprise of a phosphate group, nitrogenous bases and ribose as its pentose sugar; RNA does NOT necessarily have complementary base -pairing of A=U, G≡C! This base -pairing only occurs in secondary RNA structures. RNA is NOT used to synthesize mRNA, tRNA and rRNA! It EXISTS as either mRNA, tRNA or rRNA. 8. Messenger RNA is a single-stranded RNA which is transcribed in the nucleus and transported into the cytoplasm for translation by ribosomes; 9. Ribosomal RNA is a single-stranded RNA synthesized in the nucleolus and assembled with ribosomal proteins to form ri