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 c