BMS1062:Molecular Biology: DNA

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Biomedical Sciences
Peter Boag

INTRO TO MOLECULAR BIOLOGY:  Genetics: the study of all aspects of genes  Genomics: the study of complete gene sets (genomes) DNA STRUCTURE AND PHYSICAL PROPERTIES:  DNA is a linear series of four molecular building blocks called nucleotides  DNA nucleotide structure: each nucleotide contains: o Deoxyribose sugar o Phosphate group o Nitrogenous base  Four different bases: Adenine-Thymine, Guanine-Cytosine o Purines: A and G (Double carbon-nitrogen ring) o Pyrimidines: T and C (U for RNA) - (Single carbon-nitrogen ring) o A-T have 2 H-bonds o G-C have 3 H-bonds o In DNA, # A = T and G = C  Bases face the centre of the double helix and each base is hydrogen bonded to the base facing it on the opposite strand.   In RNA the ribose sugar is part of the nucleotide (not the deoxyribose sugar) Genome: organisms complete set of genetic information, encoded in its DNA Genes: segments along one continuous DNA molecule and are the primary information carriers in the genome Diploid: organisms nucleus contain two identical chromosome sets (2n) Haploid: number of chromosomes in the basic genome set; contain one chromosome set (n) Two members of a chromosome pair are called Homologous chromosomes or ‘homologs’  DNA molecules in a genome can be separated by size on a gel using electrophoresis. o The number of DNA bands observed on the gel is equal to the Haploid chromosome number confirming that each chromosome contains only one DNA molecule. DNA packaging: Coiling the DNA double helix around molecular spools called nucleosomes. o Each nucleosome is composed of eight proteins called histones. o The DNA and associated nucleosomes are together called chromatin. Chromosomes:  Tip of the chromosome = telomere.  Centromere: attachment point to move the chromosome during cell division. Linkage of nucleotides:  5’ phosphate (C5) – 3’ OH (C3)  Phosphodiester bond (covalent)  Catalysed by a polymerase Primary structure of DNA  Linear sequence of bases in a single strands (hydrophobic)  Always read 5’-3’  Sugar phosphate backbone (high –ve charge) RNA:  4 Bases – A,U,G,C  Only one nucleotide strand  Shorter than DNA Discoveries:  James Watson and Francis Crick: DNA is a double helix composed of two nucleotide strands that wind around each other  Frederick Griffith: Mice experiment with R and S strain  Oswald Avery, Collin Macleod, Maclyn McCarty (1944): chemically destroyed all the major categories of chemicals in an extract of dead cells and found out if the extract had lost the ability to transform. Mixture lost its transforming ability when enzyme Deoxyribonuclease (DNAse) was used, implicating DNA as the genetic material.  Hershey Chase: experiment that made use of phage T2, a virus that infects bacteria. Chargaff’s Rules of base composition: 1- The total amount of purine nucleotides (A + G) always equals the total amount of pyrimidine nucleotides (C + T) 2- Amount of T equals the amount of A and amount of C equals amount of G (A=T, G=C, A+T does not equal C+G) Base + Sugar = nucleoside Base + sugar + phosphate = nucleotide DNA Replication: Watson and Crick referred to ‘semi-conservative’ replication Each of the two single strands acts as a template In semiconservative replication: double helix of each daughter DNA molecule contains one strand from the original and one newly synthesised strand In Conservative replication: the parent DNA molecule is conserved and a single daughter double helix is produced consisting of two newly synthesised strands In dispersive replication: daughter molecules consists of strands each containing segments of both parental DNA and newly synthesised DNA Watson and Crick’s model Properties of DNA: 1. Solubility: soluble in aqueous / low salt conditions but insoluble in alcohol 2. Viscosity: very viscous (disruption of H-bonding decreases viscosity; i.e. dsDNAssDNA) 3. UV Absorption: nitrogenous bases strongly absorb UV light: ssDNA absorption > dsDNA 4. Denaturation: H-bonding is disrupted, caused by increased temperature, extremes of pH etc. - decreases viscosity, increases UV absorption 5. Stability: Tm is a characteristic of DNA that depends on the GC content. Tm ∞ G + C More energy is required to break GC pairs than AT pairs Hybridisation:  Two DNA species are heat denatured, mixed and cooled  In addition to renaturation, hybrid molecules will form where the species have complimentary sequences.  DNA-RNA hybrids are possible Probes: labelled (usually radioactively) DNA or RNA fragments used to detect specific sequences by hybridisation. RNA polymerase has no proof reading activity and thus makes more errors than DNA polymerase which has proof reading ability Bacterial genome organisation:  Generally single circular chromosome  Some have two circular chromosomes  Some have linear chromosomes Plasmids: autonomously replicating pieces of double stranded DNA  Can be present in many copies (unlike chromosomes: single copy)  Copy number is the average # copies of a plasmid in a cell  Once cellular replication occurs, the plasmid count will be reduced, however the plasmid will rapidly attempt to keep the copy number constant and will grow until
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