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BIOL 205 (111)


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Queen's University
BIOL 205
Ian D Chin- Sang

BIOL 205:week 10, lecture 28 How to monitor mRNAexpression level for every gene: Global transcriptional Profiling • carry out thousands Northern Blots? • instead – DNAmicroarrays were developed • DNAmicroarrays for global transcriptional profiling were not feasible before the sequencing of whole genomes ◦ had to make an array to each sequence of DNAspecific to each mRNA • Northern Blots: immmobilized mRNA population hybridized with labeled DNAprob representing one or two genes • DNA Microarrays: immobilized DNAprobes representing all possible genes hybridized with labeled mRNApopulation Need to achieve two things: (i) Immobilize (array) thousands of DNAprobes specific for each individual mRNA gene product (ii) Label mRNA populations → up to 44,000 probes per slide Robots designed to spot 44,000 Individual DNA spots The probes canbe cDNAs (~1Kb) or oligonucleotides (20-70 mers) → advantage to oligonucleotides: chemically synthesize and get exact nt sequence **CautionaryNote:May miss extremelyunstable mRNAs Microarrays and Spot Colour Equal More More Insufficient amnts green red hybridization arrayof1046 cDNAs AffymetrixGene chip probed withfluorescently 65,000 oligonucleotide labelled cDNA arrayrepresenting1641 made frombone marrow genes mRNA Canblock proteinsynthesis withcycloheximide → tells youwhether youneed new proteinsynthesis to Eachrow is a different change transcript level gene, eachcolumna different time point increase Red indicates mRNA transcript levels are higher Greenindicates mRNA transcript levels are lower Should we carrya microarray credit card? - personalized medicine:diagnose changes, predict disease or infection - predict drugefficacyor toxicityin individuals → problems:ifinsurance companies for info that youhave mRNAfor certaindisease and deny insurance → Hard to knockout genes inmammaliancells RNAi –Opportunity for global gene knockout! → Introducing double stranded RNA inhibits the respective gene. Ex:ifchose actingene 1. Degrades RNA And made dsRNA:goingto 2. Blocks translation 1 knock out allactingenes 3. Recruits chromatinremodelers to block further transcription Dicercleave into 2 exactly 21nucleutides silencing RNAs picked up 3 by... RNAinduced silencingcomplex 4 MRNAthencleaved 5 → now people are tryingknockouts ofeverydifferent gene and analyzingfunction Functional genomics using high-throughput RNA interference C. elegans has a Lookingfor ways to knock mechanisms that moves out viralRNA the RNAifromcellto cell untileachcellexpresses RNAi Polymerase Chain Reaction -Atechnique to amplify DNA • History controversial: • process conceptualized by Har Gobind Khorana in a • paper published in 1968 • Mullis refined the process in 1983 while working at • Cetus (company) – came up with temperature cycling • 1986 –introduced the use of a thermostable polymerase (Taq) from Thermophilus aquaticus • Cetus gave him a $10,000 bonus – sold the patent for $300 million years later • 1992 – Mullis formed a company to sell amplified DNAof Elvis Presley and Marilyn Monroe in amber jewelry. • later promotedAIDS and climate change denialism • claims he could not have made his discovery without LSD use Requirements for PCR • DNAtemplate –sample DNAthat contains the target sequence – high heat (95-100C) applied to separate strands • primers –short pieces (generally 18-24 nctds of single stranded DNA- complementary to the target sequence • Deoxynucleotide triphosphates (dNTPs) • Buffers and salts • Athermostable polymerase – Taq or Pfu Where do thermostable polymerases come from ? • in 1966, Thomas Brock made the remarkable discovery that microorganisms were growing in the boiling hot springs of Yellowstone National Park • "Thermophiles" are microorganisms with optimal growth temperatures between 60 and 108 degrees Celsius, isolated from a number of marine and terrestrial geothermally-heated habitats • including shallow terrestrial hot springs, hydrothermal vent systems, sediment from volcanic islands, and deep sea hydrothermal vents • Taq polymerase isolated from Thermis aquaticus was the first and most frequently used ◦ not proofreading • Pfu polymerase (Pyrococcus furiosus) has greater fidelity (has proofreading: 3'-5' exonucleases) • there are now numerous heat stable polymerases available • commerciall
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