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Lecture 10

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BIOL 205

How to monitor mRNA expression level for every gene: Global transcriptional Profiling • Carry out thousands Northern Blots? • Instead – DNA microarrays were developed • DNA microarrays for global transcriptional profiling were not feasible before the sequencing of whole genomes. Northern Blots Immobilized mRNA population hybridized with labeled DNA probe representing one or two genes DNA Microarrays Immobilized DNA probes representing all possible genes hybridized with labeled mRNA population Need to achieve two things: (i) Immobilize (array) thousands of DNA probes 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 can be cDNAs (~ 1Kb) or oligonucleotides (20-70 mers) 4x44K spots “features” US/products/ instruments/dnamicroarrays/pages/gp55 mRNA mRNA population 1 population 2 Cautionary Note: May miss extremely unstable mRNAs RT/PCR Label with fluorescent dye Mix equal PROBE [ ] amounts In excess Hybridize to array wash Monitor Fluorescence for each Fluorescent dye Affymetrix Gene chip Array of 1046 cDNAs probed with fluorescently labelled cDNA 65,000 oligonucleotide array made from bone marrow mRNA Representing 1641 genes Can block protein synthesis with cycloheximide -tells you whether you need new protein synthesis to Each row is a different change transcript level gene, each column a different time point Red indicates mRNA transcript Levels are higher Green indicates mRNA transcript Levels lower. Should we carry a microarray credit card? •Personalized medicine: diagnose changes, predict disease or infection •predict drug efficacy or toxicity in individuals RNAi –Opportunity for global gene knockout! Introducing ds RNA inhibits the respective gene. RNA induced Silencing complex 1. Degrades RNA 2. Blocks translation 3. Recruits chromatin further transcription # of RNAi based drugs COMPANY Polymerase Chain Reaction -A technique 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 – came up with temperature cycling Kary Mullis • 1986 –introduced the use of a thermostable Nobel in polymerase (Taq) from Thermophilus aquaticus • Cetus gave him a $10,000 bonus – sold the patent for Chemistry 1993 $300 million years later • 1992 – Mullis formed a company to sell amplified DNA of Elvis Presley and Marilyn Monroe in amber jewelry. • Later promoted AIDS and climate change denialism • Claims he could not have made his discovery without LSD use. Requirements for PCR • DNA template –sample DNA that 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 • A thermostable 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 Pfu polymerase (Pyrococcus furiosus) has greater fidelity. There are now numerous heat stable polymerases available Commercially – hot tub, Vent, Deep Vent The Basics of PCR Cycling • 30-35 cycles each comprising: - Denaturation (95 C) – 3
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