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17 Lec 17 Molecular Methods Part 2

17 Lec 17 Molecular Methods Part 2 - Molecular analysis of...

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Molecular analysis of soil microbial communities II. Function of soil microbial communities Eoin Brodie, LBNL [email protected]
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Summary Who’s there? – Biomarkers: DNA extraction » PCR amplification » clone libraries / microarrays How many? – Relative abundance: Clones / microarray – Actual abundance: qPCR / FISH
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Today Connecting identity (phylogeny) to function qPCR, qRT-PCR Functional gene microarrays Metagenomics Sequencing technology Sanger 2nd generation Determining function Soil systems biology Metatranscriptomics/proteomics (Stable) Isotope probing
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Monitoring functions • Biochemistry – Determine in situ enzyme activity/potential Fluorogenically-labeled substrates – E.g. methylumbelliferyl (MUB) conjugated substrates – Enzyme activity results in fluorescent signal – No identity of organisms carrying out function
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Phylogeny = Function? Sometimes it works Nitrosomonas species are ammonia oxidizers They make their living doing this, so their activity means that ammonia oxidation must be going on Methanococcus species are methanogens Activity of Methanococcus indicates methane production Nitrate reduction or Iron reduction Capability found in many bacterial (and some archaeal) phyla - not specific to any groups - many of these can organisms can also use alternate electron acceptors
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Monitoring functions Functional gene presence (DNA) / activity (RNA) Nitrogen cycle Ammonia oxidation ammonia monooxygenase (amoA) • Denitrification nitrate reductase (narG), nitrite reductase (nirK, nirS), nitrous oxide reductase (nosZ) Carbon cycle Cellulose - cellobiohydralase (cbh2) Hemicellulose - betaxylosidase (bxl1) Lignin - lignin peroxidase (lip) Chitin - chitinase (chiA) Methanogenesis - methyl coenzyme-M reductase (mcrA) Aerobic methane oxidation - methane monooxygenase (mmo)
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Known functional genes Low number of targets – Cloning-Sequencing – qPCR (DNA), – qRT-PCR (mRNA) High number of targets – Microarrays (DNA/RNA)
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Known functional genes Microarrays - GeoChip (He et al., 2007)
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Undiscovered genes DNA from an entire community – Metagenome(ics) (Handelsman, 1998) "the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species.” Kevin Chen and Lior Pachter (UCB) – Enabled by the Human Genome Project (2003) Massive increase in DNA sequencing capacity Specialized centers Joint Genome Institute (JGI) The Institute of Genome Research (TIGR) The Broad Institute (Harvard/MIT - formerly Whitehead) Washington University St. Louis Baylor College
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Metagenomics 1998 - Handelsman - (term metagenomics first used) 2000- Beja/DeLong - Marine water (DNA fragments, 150 x10 3 bp) 2004 -Tyson/Banfield - Acid mine (near complete genomes, 76 x 10 6 bp) 2004 - Venter - Saragasso Sea - (genes/fragments, 1 x 10 9 bp - 1.2 x 10 6 new genes)
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