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Nucleic Acids, DNA Replication, Transcription, Translation and Application to Molecular Detection Prokaryotic Cell Binary Fission

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Nucleic Acids, DNA Replication, Transcription, Translation and Application to Molecular Detection Prokaryotic Cell Binary Fission Fundamentals: Nucleic acids, DNA replication, transcription, translation and application to molecular detection Prokaryotic cell Binary Fission • Bacteria reproduce asexually via binary fission • Each daughter cell is an identical copy (or clone) of its parent cell Microbial evolution 101 Generation 1 Ancestor Genotype Generation 2 Clones Generation 3 Clones Clones and Generation N Divergent Genotypes Microbial genetics 101 • What is DNA ? • What types of DNA molecules are present in a bacterial cell? • What’s the size of the genetic material for a typical bacterial pathogen ? • How many genes does a bacterial pathogen have ? • What’s the average size of a bacterial gene ? What Is DNA? Double Helix Structure and Anti- parallel Orientation Nucleotide = 5 carbon sugar + nitrogenous base + phosphate group DNA is a polynucleotide Constituents of a Gene • Promoter • Ribosome binding site • Open reading frame • Start & Stop codons Start Stop codon codon ATG TAG 5’ 3’ -35 -10 Promoter Ribosome binding site The Central Dogma DNA DNA replication Molecular Transcription methods mRNA Translation Protein/Enzymes Classical methods Post-translation Toxins & other metabolites DNA Replication • Topoisomerases remove superhelicity • New DNA is synthesized in the 5’ to 3’ direction • Replication begins at a the origin of replication (ori) • Two replication forks proceed around the chromosome (bi-directional) until they encounter termination (ter) sites • Replication is continuous on one strand and discontinuous on the other strand • Chromosomes partitioned into two daughter cells during cell division DNA Replication Leading strand; Lagging strand; continuous discontinuous replication replication • Helicase; unwinds helix • Single-stranded binding protein; binds single-stranded DNA prevent hybridization • Primase; lays down RNA primers needed for DNA polymerase activity • DNA polymerase I; remove RNA primers replace with DNA • DNA polymerase II; DNA repair • DNA polymerase III; major replication enzyme forms phosphodiester bonds • Ligase; seals nicks by linking free 3’ OH with 5’ adjacent phosphate group • Proofreading (DNA pol I & III) 3’ to 5’ exonuclease activity to remove incorrect base • Incorrect base incorporated every 1x108 to 1x1011 bases DNA Polymerase and PCR • DNA polymerase III • Polymerase with or without 3’ to 5’ exonuclease proofreading activity – Taq (5’ - 3’ exonuclease activity); only degrades double stranded DNA while extending – Vent (3’ to 5’ exonuclease activity; specialized Taq) – Implications • Detection/subtyping methods • Cloning DNA Replication and Application to Molecular Detection • Polymerase chain reaction (PCR) or “DNA photocopying” – Simulate the natural DNA replication process to make copies of DNA in vitro – Make many copies of specific DNA fragment(s) in vitro • Template, deoxynucleotidetriphosphates, primers, DNA polymerase, enzyme cofactors, and buffer RNA v. DNA RNA in the cell • Ribosomal RNA (rRNA) bulk of RNA in a cell – 3 types (16s, 23s, and 5s) – 3,000 copies in a cell – Ribosomes; protein assembly during translation • Messenger RNA (mRNA) 5-10% of RNA in a cell – Almost as many types as there are genes – Not stable in the cell; highly transcribed genes have a few hundred copies; half-life a few minutes (1 to 7 min.) – Synthesized from DNA during transcription – Move information contained in DNA to translation machinery • Transfer RNA (tRNA) – About 50 types – Pick up amino acid & transport to ribosome during translation • Small RNA (sRNAs) – 50 - 200 nucleotides – Regulatory roles (e.g., affect mRNA stability and translation) The Central Dogma DNA DNA replication Molecular Transcription methods mRNA Translation Protein/Enzymes Classical methods Post-translation Toxins & other metabolites Transcription and translation are coupled in bacteria Holoenzyme (RNA polymerase and sigma factor 5’ 3’ 3’ Transcription 5’ tRNA Anticodon mRNA 50S large subunit (23S and Ribosomes 5S RNA and Translation proteins) Direction 30S small subunit (16S Translated RNA and Protein proteins) A closer look at transcription • DNA used as template to synthesize complementary mRNA molecules • RNA polymerase (pol) binds to promoter region in double-stranded DNA • Sigma factors help RNA pol bind promoter & target genes to be transcribed • -10 and -35 region 5’ of transcription start site • Local unwinding of double-stranded DNA • RNA pol recognizes transcription start site • RNA pol adds nucleotides 5’ to 3’ • RNA pol termination RNA pol and RNA molecule released •Rho-dependent •Rho-independent (hairpin loop; termination sequence) The Central Dogma DNA DNA replication Molecular Transcription methods mRNA Translation Protein/Enzymes Classical methods Post-translation Toxins & other metabolites A closer look at translation • Three ribosome sites: • A site; entry of aminoacyl tRNA (except 1st aminoacyl tRNA or start codon, which enters at P site) • P site; peptidyl tRNA is formed • E site; exit for uncharged tRNA • Shine-Dalgarno sequence or ribosome binding site recognized (5-10 bases upstream of start codon) • Assembly of small and large ribosome subunits • Amino acids added to carboxyl end of growing chain • Protein exits ribosome through tunnel in large subunit • Termination occurs when one of three termination codons moves into A site Genetic code Application to molecular detection in food microbiology • Molecular detection methods include assays that target nucleic acids (i.e., DNA and RNA) • DNA detection methods – Detect presence or absence of gene(s) or gene fragment(s) specific to the target organism – Detection of universal gene or gene fragment (e.g., 16s rRNA) followed by DNA sequencing – Detection of DNA does not differentiate between viable and non- viable organism • mRNA detection methods – mRNA is rapidly degraded and detection indicates presence of viable organism PCR Applications • PCR detection particularly useful when – Classical detection too time-consuming – Differentiation from closely related non-pathogenic organisms is difficult • Listeria monocytogenes – Only species in Listeria genera that is pathogenic to humans – PCR assay targeted to detect hemolysin (hlyA) gene can detect presence and differentiate L. monocytogenes from other Listeria spp. PCR Reaction Components • 1 - Small quantity of DNA added to tube • 2 - DNA polymerase • 3 - Oligonucleotides (primers) • 4 - Deoxynucleotidetriphosphate bases • 5 - MgCl2 • 6 - Buffer • 7 - Sterile ultrapure water Polymerase Chain Reaction (PCR) Fundamentals DNA Extraction Introduction to PCR • DNA Genetic information for every animal, plant and microorganism • Unique variations in DNA allow us to track it back to the organism it originated from with precision • Comparative genomics, forensics, fingerprinting often require significant amounts of DNA • PCR can synthesize, characterize and analyze any specific piece of DNA What is PCR ? Polymerase Chain Reaction: – in vitro (DNA synthesis in a tube) – Yields million of copies of target DNA sequence – Repeated cycling action – Involving DNA polymerase enzyme PCR Principles • Conceptualized by Kary Mullis in 1983 • DNA amplification in vitro using the following components: – Two synthetic oligonucleotides (primers) complementary to each end of targeted DNA sequence – Single nucleotide bases as substrate – DNA polymerase; a naturally occurring enzyme responsible for in vivo DNA replication and repair PCR Applications • Food Science: – Detection or molecular confirmation of specific microorganisms present in foods – Molecular subtyping of isolates • Molecular Biology: – Mutagenesis, cloning or sequencing • Evolutionary Biology: – Re-create the evolutionary history of a group of taxa PCR Applications • PCR detection particularly useful when – Classical detection too time-consuming – Differentiation from closely related non-pathogenic organisms is difficult • Listeria monocytogenes – Only species in Listeria genera that is pathogenic to humans – PCR assay targeted to detect hemolysin (hlyA) gene can detect presence and differentiate L. monocytogenes from other Listeria spp. PCR Reaction • High temperature “melts” double strand DNA helix into single strand DNA • Two synthetic sequences of single stranded DNA (18-24 bases) known as primers target a region of genome • Forward primer and Reverse primer flank the region of interest (usually <1000 base pair) PCR Reaction Components 1 – DNA template 2 – DNA polymerase 3 – Primers (oligonucleotides) 4 – Deoxynucleotidetriphosphate bases (dNTPs) 5 – MgCl2 6 – Buffer 7 – Sterile ultrapure water PCR Reaction Set-up 1 - DNA Template – Theoretically, PCR can detect as little as one DNA molecule – Template DNA should be present in small amounts (< 106 target molecules; 1 ng of E. coli DNA = 3x105) – Bacterial cells need to be lysed to make their DNA accessible for PCR PCR Reaction Set-up 2 – Primers – Should be in great excess to template DNA to ensure that most strands anneal to a primer and not each other – Generally between 0.1 and 0.5 µM optimal concentration – Higher primer concentrations may cause non- specific products PCR Reaction Set-up 3 - Deoxynucleotide Triphosphates (dNTPs) – Building blocks of DNA A’s, T’s, G’s, & C’s – Essential to have enough dNTPs to make desired number of target sequence copies – Final concentration of EACH dATP, dTTP, dCTP and dGTP should be 0.1 mM PCR Reaction Set-up 4 - DNA Polymerase Enzyme: – Original PCR performed with E. coli DNA polymerase, but high temperature (94-95oC) needed to denature double stranded DNA also denatured this polymerase
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