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This Week: Mon—Omics Wed—Alternate sequencing Technologies and Viromics paper Next Week No class Mon or Wed Fri– Presentations by Colleen D and Vaughn Discussion of Environmental Community Phylogeny paper (led by John) Finals week Monday 9-noon Presentations by rest of class Wed 5pm—Final Papers Due Other Kinds of “Omics” Why would we want to assay RNA and proteins on a genome wide scale in the environment? Can we assay RNA and proteins on a genome wide scale in the environment? RNA----sequencing --detection via hybridization Proteins—separation and identification RNA Detection : Reverse Transcription Goal: RNA DNA Ingredients: RNA (total, or poly A) Enzyme—reverse transcriptase (viral in origin) Nucleotides Primers—poly T or random hexamers Reverse Transcription 5’ 3’ A T C A A G G G C A CG T C T RNA TTTTTTTT or N6mers Reverse Transcriptase Result is a pool of single stranded DNA Complementary to RNA (cDNA) 3’ 5’ Reverse Transcription cDNA can then be used as a template in PCR, using specific primers for gene of interest (RTPCR) cDNA could be cloned to form a cDNA library cDNA can also be stored for future experiments—more stable than original RNA Issues with Reverse Transcription Too much rRNA is problem for prokaryotes Often results in incomplete fragments; Heavily 3’ biased in eukaryotes Environmental Transcriptomics 400 clones from Georgia Salt Marsh Identities of cDNA fragments 16S rRNA clone library Poretsky et al 2005 Global Transcriptional Profiling: Microarrays Principle is similar to Northern Blot Northern Blot: total RNA separated by size 1 gene is labeled-probe Signal indicates hybridization-> expression Microarray: like doing thousands of Northerns simultaneously Thousands of known genes separated by space Total mRNA (or cDNA) is labeled Signal indicates hybridization-> expression Northern Blots Isolate RNA Run on gel to separate by size Hybridize with labeled probe Transfer to membrane Wash and detect Examining gene expression using DNA microarrays Step 1---Construct gene chip From whole genome sequence or sequenced cDNA library either: PCR each individual gene or synthesize 70mer specific for each gene PCR products or 70mers are physically spotted onto a glass substrate (using a robot) Each spot represents a single gene Examining gene expression using DNA microarrays Alternative Step 1---Purchase gene chip From whole genome sequence or sequenced cDNA library Design short oligos (1522mers) tiled along the length of the genome/clones Chip with desired oligos is commercially synthesized Several spots for each gene Step 2 ---the experiment Test condition Isolate mRNA RT to cDNA and label Pool apply to chip Fluorescence detection Reference condition Observe relative changes in gene expression Microarrays--issues $$$$$ both to synthesize the chips (ordering thousands of primers or 70mers) and to buy the dyes to label the cDNA for each experiment Genes should be spotted in duplicate or triplicate Need to do reverse label experiments to confirm results reference sample biological issues statistical issues Sensitivity—highly dependent on background Total amount of mRNA needed can be high (esp. for prokaryotes) May not be quantitative—genes of particular interest often confirmed to be differentially expressed via Northern blot or RT-PCR Microarrays—how to take into environment? Current environmental approaches tend to be taxonomic arrays: Many versions of a single gene (16S rRNA, nifH, amoA, nirS, nirK) spotted on the array Hybridization shows which types are expressing gene in sample NifH macroarray Jenkins et al 2004 NifH macroarray shows expression of different types of nifH in the Chesapeake Bay Jenkins et al 2004 Proteomics Proteins have very different properties than nucleic acids Cellular localization Have 3d structure (active and inactive forms) Size, charge, hyrdrophobicity are different from NAs and from each other One principle is similar—in order to identify them from a mixture, need methods of separation Proteins Two types of electrophoresis SDS-PAGE = denaturing Polyacrylamide Gel Electrophoresis Non-denaturing: Preserves native protein conformation and activity Denaturing reducing agents (urea) or detergent (SDS) used to break intramolecular bonds and linearize protein, and impart uniform negative charge useful for determining size From Sigma total protein plant extraction kit Each amino acid has a characteristic pI (isoelectric point) the pH at which it carries no charge Combination of amino acid content and 2º and 3º strcuture give each protein a pI Proteins---2 dimensional gels Stage 1Isoelectric focusing — separates by native charge Proteins---2 dimensional gels Stage 2----SDS-PAGE separates by size Often used to compare two different conditions/treatments to identify proteins unique to 1 condition Check out http://us.expasy.org/ for proteomics/2D gel data resources Protein ID is heavily dependent on Database of potential peptides Proteomics—can be quantitative