BIOL1020 Module 2 - Molecular Genetics

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Dr Paul Ebert

Module 2: Molecular Genetics DNA as Genetic material DNA exists as a helix of two antiparallel strands which interact through weak hydrogen bonds  Nucleotides linked by phosphodiester bonds  Have direction due to 3’ and 5’  5’ end has a phosphate group where 3’ has an OH group DNA comprised of three groups  Base  Sugar  Phosphate DNA consists of four subunits: A,C, G & T  Adenine pairs with thymine  Guanine pairs with cytosine Adenine and guanine = purines (two carbon-nitrogen rings) Cytosine, thymine and uracil = pyrimidines (one carbon-nitrogen ring) Sugars in DNA and RNA Both sugars are pentose sugars with 5 carbon atoms DNA: beta-D-2-deoxyribose RNA: beta-D-ribose Packing of DNA DNA viruses typically consist of >10,000 nucleotides Human chromosome may contain 50 to 250 million base pairs  To fit into nucleus the chromosome must be 10,000 times smaller DNA associated with basic (positively charged) proteins (the histones) which form protein-DNA complexes known as nucleosomes  Contains 200 nucleotides with other histones (proteins) (linked by Histone H1) - Interaction between histones’ positive charge and negative charge of DNA  Wound into solenoid-like structure with 6 nucleosomes per turn  Forms 30nm chromatin fibre  Chromatin fibre packed into loops attached at intervals to a scaffold of protein Models for DNA replication Semiconservative model 1. Parent strand separates into two and each create a replicant 2. Two daughter molecules consist of one parental strand and one new strand 3. After second replication, two of the DNA molecules consist only of new material, while other two consist of one new and one old Conservative model 1. Parent strand directs synthesis of an entirely new double-stranded molecule 2. After replication, one molecule is conserved as two old strands Dispersive model 1. Parent strand material is distributed randomly between daughter cells DNA Replication complex Eukaryotic DNA is replicated at ~50 nucleotides/second 1. Helicase unwinds the parental double helix to single strands 2. Molecules of single-strand binding protein stabilize unwound template strands 3. Leading strand is synthesized continuously in the 5’ to 3’ direction by DNA polymerase III 4. Primase begins synthesis of RNA primer for fifth Okazaki fragment 5. DNA pol III completes synthesis of 4 fragment and reaches RNA primer on the third fragment and dissociates 6. DNA pol III moves to replication fork and adds DNA nucleotides to the 3’ end of the fifth fragment primer 7. Leading strand synthesized continuously in the 5’ to 3’ direction by DNA polymerase III Components of replication  Template DNA: plasmid or PCR template to replicate  Single primer (oligonucleotide)  Taq DNA polymerase: to incorporate polymerase  Deoxyribonucleotide triphosphate: to be incorporated into the synthesized DNA  Dideoxyribonucleotide triphosphates: dye terminators to stop reaction  Buffer: creates environment for reaction DNA topoisomerase: prevents supercoiling by transiently nicking both strands and allowing two strands to rotate around each other DNA polymerase: catalyse addition of deoxyribonucleoside triphosphate to the 3’-OH end of DNA RNA primer: removed by action of the 5’ exonuclease activity of DNA polymerase I DNA synthesis on the lagging strand is discontinuous, forming short DNA strands (Okazaki fragments) DNA proofreading and repair Mechanisms used to avoid mutation Nucleotide excision repair removes thymine dimers caused by UV radiation PCR and DNA amplification Polymerase Chain Reaction (PCR): controlled in vitro enzymatic reaction that mimics the DNA replication process to replicate a specific piece of DNA Enzymatic amplification first proposed in early 1970s  Discovery of thermus aquaticus – heat- resistant replication enzymes of this organism can tolerate repeated cycles of denaturation required for PCR to work  Used in: - Used in DNA fingerprinting - Recovery of ancient DNA samples from archaeological sites - Gene cloning and construction of synthetic genes DNA Electrophoresis Utilises a polymer matrix constructed from agarose or acrlamide Ability to separate DNA fragments based on size  Negatively charged DNA molecules migrate through a gel in an electric field DNA sequencing Sanger dideoxy terminator: uses labeled dideoxy derivatives of nucleotide triphosphates that block extension of growing DNA chain  Labelled with different coloured dye to distinguish the terminator base Gene sequencing Genes = software for cells  Genes are ‘expressed’ to determine phenotype i.e. development, behaviour
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