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