Transcript Lecture

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