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Spafford BIOL 208 Notes

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BIOL 309
David Spafford

University of Waterloo Department of Biology BIOL 208 – Analytical Methods of Molecular Biology CHAPTER 2: BASIC MOLECULAR BIOLOGY 2.1 NUCLEIC ACID STRUCTURE 2.1.1 The DNA Backbone - Phosphate-sugar backbone - 5’ P links one sugar to 3’ position of next Degradation of DNA require nucleases that break covalent bonds… - Endonuclease – cut internal sites - Exonuclease – nibble ends Charateristics of RNA: - Contains ribose - Less stable than DNA - Greater susceptibility to degrade by ribonucleases chemical degradation by alkaline conditions 2.1.2 The Base Pairs - Purines: Adenine and Guanine (larger) - Pyrimidines: Thymine and Cytosine (smaller) Figure 1. Purines vs. Pyrimidines - Strands are antiparallel – running 5’ (phosphate) to 3’ (hydroxyl) in either direction - DNA easily denatured – H bond breaks without affecting sugar-P - Easily renature – complementary base pair Melting Temperature for DNA - GC (3 H bonds) melt less easily than AT (2 H bonds) - Estimate T m> known sequence and buffer conditions - DNA structure maintained by hydrophobic interactions between bases - ssDNA/RNA unstable in water - Pairing of bases removes instability Importance of Hydrophobicity in Hybridization Secondary structures of ssDNA/RNA: hairpins, stem-loop difficult to separate strands plays role in gene expression initiation of DNA replication Importance of Salt in DNA Hybridization - Negative charge on DNA backbone causes electrostatic repulsion - Presence of salt neutralizes (-) charge on P groups - Reduction of [salt] – weak interactions between strands Low salt cond. increase specificity of hybridization 2.1.3 RNA Structure - Ribose and less stable - Easily destroyed by high pH - Uracil rather than Thymine - Most are single-stranded 2.1.4 Nucleic Acid Synthesis - Nucleotides always added to growing end (3’ OH) - Substrate is dNTP in DNA and NTP in RNA - DNA needs primer to get started 2.1.5 Coiling and Supercoiling B-DNA - Standard, relaxed form - Right-handed double helix - One turn per 10 bp A-DNA - Double helix - Right-handed but more compact - One turn per 11 bp Z-DNA - Left-handed double helix - Irregular zigzag structure Types of Circular Forms (a) Closed Circular Form - Naturally supercoiled plasmid DNA - Supercoiling occurs with DNA bearing fixed end - Amount of supercoiling influences degree of coiling - Plasmid DNA constrained in vivo (b) Open Circular Form (B Form) - DNA relaxes after breakage of plasmid - DNA remains in B form - Reform circular plasmid in vitro - Topoisomerase (DNA gyrase) breaks DNA strands, unwinds DNA and reseals 2.2 WHAT IS A GENE? Gene: a portion of the genome Imprecise definition for gene: - Refers to unit of inheritance of observable characteristics (phenotype) - Open reading frame (ORF) – region between start and stop codons 2.3 INFORMATION FLOW: GENE EXPRESSION - DNA fundamental genetic material - RNA is final product of gene 2.3.1 Transcription - Carried out by DNA-dependent RNA polymerase II - Polymerase I and III make rRNA and tRNA - RNAP binds to promoter sequence – initiates synthesis of mRNA (adjacent) - Bacterial promoter carrier two consensus sequences at position -35 and -10 - RNAP – higher affinity Trans-acting transcription factors: DNA binding proteins encoded by genes in other parts of genome Cis-acting promoter elements: elements that affect expression of gene next to them - Nucleotide cap added to 5’ end -> serves a site recognized by ribosome’s - Pre-cursor mRNA cleaved at 3’ end and poly-A tail added to cut end - Polyadenylation recognition sequences in 3’ untranslated region - Introns spliced out -> exons joined together 2.3.2 Translation - Starts when ribosomes bind to ribosome binding site (RBS) (adjacent to start codon) - RBS is Shine-Dalgarno Sequence (complementary to 3’ end of 16s rRNA - Ribosomes bind at 5’ end of mRNA to cap 2.4 GENE STRUCTURE AND ORGANIZATION 2.4.1 Operons - Group of genes (or operon) transcribed from single promoter into one long RNA - Operons associated in same regulatory pathway 2.4.2 Exons and Introns Eukaryotic genes contain introns -> removed from mRNA transcript before translation Introns infrequent in bacteria: - Bacteria genomes small and evolutionary pressures keep them small - Ribosomes translate mRNA while it’s being made CHAPTER 3: HOW TO CLONE A GENE 3.1 WHAT IS CLONING? Cloning: using asexual reproduction to obtain genetically identical organisms - Cloning extends to genes: introduction of foreign gene into bacterium - Cloned genes identical in all replicating bacteria harboring that gene 3.2 OVERVIEW OF THE PROCEDURES - Bacteria take up DNA by transformation when subject to Ca and heat (heat shock) or electrical pulse (electroporation) - DNA only replicated if inserted into host chromosome or in vectors recognized by enzymes within host cell as subtrate for replication Plasmids or bacteriophage: bacterial vectors Figure 2. Basic Outline of Gene Cloning - DNA insert cloned into vector produce recombinant DNA Replicated in all descendants of initial transformant - Enzymes restriction endonucleases break DNA sugar-P backbone - Cut insert and vector producing sticky or blunt ends - Insert and vector joined with DNA ligase - Small fraction of ligated insert and vector taken up by bacteria during transformation 3.3 GENE LIBRARIES - Specific gene isolated from genome using genomic libraries - Isolated genomic DNA cut into DNA fragments with restriction enzymes - Mixture of DNA fragments ligated into vectors - Colony transformed from mixture DNA containing only one DNA fragment - Each bacterial colony inoculated into fresh media Figure 3. Making a Genomic Library From Small Genome 3.4 HYBRIDIZATION - Common technique to identify one gene matching specific DNA sequence (DNA probe) - Heating denature/melt gene library fragments + DNA probe - Upon cooling, DNAs reanneal - Specificity of hybridization depends on stringency of hybridization 3.5 POLYMERASE CHAIN REACTION Provides alternative to gene cloning and gene libraries Obtain usable quantities of specific DNA sequences - Requires pair of primers that will anneal to sites at either side of DNA - DNA poly
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