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This Week: Mon—OmicsWed—Alternate sequencing Technologies and
Viromics paper
Next Week No class Mon or WedFri– Presentations by Colleen D and VaughnDiscussion of Environmental Community
Phylogeny paper (led by John)
Finals weekMonday 9-noon Presentations by rest of classWed 5pm—Final Papers Due
Other Kinds of “Omics”
Can we assay RNA and proteins on a genome wide scale in the environment?
RNA----sequencing--detection via hybridization
Proteins—separation and identification
Why would we want to assay RNA and proteins on a genome wide scale in the environment?
Goal: RNA DNA
Ingredients:RNA (total, or poly A) Enzyme—reverse transcriptase (viral in origin)NucleotidesPrimers—poly T or random hexamers
RNA Detection : Reverse Transcription
Reverse Transcription
GG
AA
CC
GG
TTAA CC
GG
TTAA
CC
TT
AA
CC
GG
Reverse Transcriptase
5’5’ 3’3’ RNA
TTTTTTTTor
N6mers
Result is a pool of single stranded DNA
Complementary to RNA (cDNA)
3’3’ 5’5’
cDNA can then be used as a template in PCR, using specific primers for gene of interest (RT-PCR)
cDNA could be cloned to form a cDNA library
cDNA can also be stored for future experiments—more stable than original RNA
Reverse Transcription
Too much rRNA is problem for prokaryotes
Often results in incomplete fragments;Heavily 3’ biased in eukaryotes
Issues with Reverse Transcription
Environmental Transcriptomics
Poretsky et al 2005
Identities of cDNA fragments 16S rRNA clone library
400 clones from Georgia Salt Marsh
Global Transcriptional Profiling: Microarrays
Principle is similar to Northern Blot
Northern Blot: total RNA separated by size 1 gene is labeled-probeSignal 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
Hybridize with labeled probe
Wash and detect
Run on gel to separate by size
Transfer to membrane
Examining gene expression using DNA microarrays
Step 1---Construct gene chip
Each spot represents a single gene
PCR products or 70mers are physically spotted onto a glass substrate
(using a robot)
From whole genome sequence or sequenced
cDNA library
either:
PCR each individual gene
or
synthesize 70mer specific for each gene
Examining gene expression using DNA microarrays
Alternative Step 1---Purchase gene chip
Several spots for each gene
From whole genome sequence or sequenced
cDNA library
Design short oligos (15-22mers) tiled along the length
of the genome/clones
Chip with desired oligos
is commercially synthesized
Step 2 ---the experiment
Test condition
Reference condition Observe relative changes
in gene expression
Isolate mRNA
apply to chip
RT to cDNA
and labelPool
Fluorescence detection
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 samplebiological issuesstatistical 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
Jenkins et al 2004
NifH macroarray
Jenkins et al 2004
NifH macroarray shows expression of different types of nifH in the Chesapeake Bay
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
Non-denaturing:Preserves native
protein conformation and activity
Denaturingreducing 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
SDS-PAGE= denaturing Polyacrylamide Gel Electrophoresis
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 1-Isoelectric focusing —separates by native charge
Proteins---2 dimensional gelsStage 2----SDS-PAGE separates by size
Often used to compare two different conditions/treatments to identify proteins unique to 1 condition
Check outhttp://us.expasy.org/ for proteomics/2D gel data resources
Protein ID is heavily dependent on Database of potential peptides
Proteomics—can be quantitative