Transcript Schematic courtesy of B. Crump Quantitative (Real Time) PCR

Molecular Methods in Microbial Ecology
Contact Info: Julie Huber
Lillie 305
x7291
[email protected]
Schedule:
21 October: Introductory Lecture, DNA extraction
23 October: Run DNA products on gel
Lecture on PCR
Prepare PCR reactions
28 October: Analyze gels from PCR
Lecture on other molecular methods
Readings: Head et al. 1998. Microbial Ecology 35: 1-21.
Day 1
• Introduction to molecular methods in
microbial ecology
• Extract DNA from Winogradsky Columns
The Challenge for Microbial Ecology
Habitat
Culturability (%)
Seawater
0.001-0.1
0.25
0.25
0.3
Freshwater
Sediments
Soil
How do you study something you can’t
grow in the lab?
From Amann et al. 1995 Microbiological Reviews
The Solution: Molecular Biology
DNA
Transcription
mRNA
Translation
Ribosome
Protein
•Are present in all cells- Bacteria, Archaea and Eukaryotes
•Are documents of evolutionary history
•Are the basis of all molecular biological techniques
Head et al. 1998
Head et al. 1998
DNA extraction from Winogradsky
Columns
DNA Extraction
1. Lyse cell membrane
a. Chemically  detergent
b. Physically  bead beating
2. Pellet cell membrane, proteins and other cell parts while DNA
stays in solution
3. Remove other inhibitors from DNA
4. Mix DNA with acid and salt  stick to filter
5. Wash filter-bound DNA several times with alcohol
6. Elute DNA off membrane with pH 8, low-salt buffer
Day 2
• Run an electrophoresis gel of the DNA
products extracted from your columns
• Learn about PCR
• Set up PCR reactions using the DNA from
your extractions and an assortment of primers
Head et al. 1998
Head et al. 1998
Basics of Gel Electrophoresis
•
The gel is a matrix (like jello with holes)
•
DNA is negatively charged- will run to positive
•
Smaller fragments run faster than larger ones
•
Gel contains Ethidium Bromide, which binds to
DNA and fluoresces when hit with UV light
(WEAR GLOVES!!!)
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What to do
• Mix 10 µl of your DNA with 2 µl loading buffer
• Load in well on gel
• I’ll load the ladder
• Run it
• Take a picture of it
Molecular Biology
DNA
Transcription
mRNA
Translation
Ribosome
Protein
•Are present in all cells- Bacteria, Archaea and Eukaryotes
•Are documents of evolutionary history
•Are the basis of all molecular biological techniques
Ribosomes
• Make proteins
• rRNA is transcribed from rDNA genes
21 different proteins
30S subunit
16S rRNA
31 different proteins
70S
Ribosome
50S subunit
23S rRNA
5S rRNA
The Star of the Show: SSU rRNA
•Everybody has it
•Contains both highly conserved and variable regions
-allows making comparisons between different organisms
over long periods of time (evolutionary history)
•Not laterally transferred between organisms
•HUGE and growing database
SSU rRNA
Universal
Tree of Life
BACTERIA
BACTERIA
You Are
Here
EUKARYA
EUKARYA
Modified from Norman Pace
ARCHAEA
ARCHAEA
Polymerase Chain Reaction (PCR)
• Rapid, inexpensive and simple way of making
millions of copies of a gene starting with very
few copies
• Does not require the use of isotopes or toxic
chemicals
• It involves preparing the sample DNA and a
master mix with primers, followed by
detecting reaction products
Every PCR contains:
• A DNA Polymerase (most common, Taq)
• Deoxynucleotide Triphosphates (A, C, T, G)
• Buffer (salt, MgCl2, etc)
• A set of primers, one Forward, one Reverse
• Template DNA
Typical PCR Profile
Temperature
Time
Action
95ºC
5 minutes
DNA Taq
polymerase
activation
35 cycles of:
95ºC
54ºC
72ºC
1 minute
1 minute
1 minute
DNA denaturization
Primer annealing
Extension creation
72ºC
10 minutes
Final extension
created
Slide courtesy of Byron Crump
Things you can optimize
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Temperature and time to activate Taq polymerase
Temperature and time to allow primer annealing
Temperature and time for extension
Concentration of reagents, especially primers,
dNTPs, and MgCl2
• Concentration of template DNA
• Number of replication cycles
• Etc…
Primers we are using
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16S Bacteria
16S Archaea
mcr Methanogens (Methyl coenzyme M reductase)
dsr Sulfate reducers (Dissimilatory bisulfite reductase
Reagent
Volume (µl) per reaction
Sterile H20
22.7
5X PCR buffer
10
dNTPs (8mM)
5
Taq polymerase (5 Units/µl)
0.3
Tube
Master mix
Target
Template
µl
# of reactions
Vol
F primer
µl
final volume
Vol
R primer
µl
Vol
µl
1
38
Sulfate reducers
Column DNA
2
dsr1F
5
dsr4R
5
2
38
Methanogens
Column DNA
2
ME1
5
ME2
5
3
38
Bacteria
Column DNA
2
8F
5
1492R
5
4
38
Archaea
Column DNA
2
20F
5
958R
5
5
38
Archaea
2
20F
5
958R
5
6
38
Nothing
+ control M.
jannaschii
- control (water)
2
20F
5
958R
5
Day 3
• Examine gels from PCR
• Learn about more molecular methods in
microbial ecology
Class DNA
10 kb
Andrea
Paliza
Jessica
Sarah
Rob
Amy
Methane Concentration (uM)
0
200
400
600
800
1000
1200
-5
Col 1 Rob
Col 2 Sarah
0
Col 4 Paliza
Col 5 Jessica
Depth (cm)
Col 7 Andrea
5
10
15
20
Col 8 Amy
Hydrogen Sulfide Concentration (mM)
0
10
20
30
40
-5
Col 1 Rob
Col 2 Sarah
0
Col 4 Paliza
Col 5 Jessica
Depth (cm)
Col 7 Andrea
5
10
15
20
Col 8 Amy
3 kb
Rob
Paliza
Amy
1 kb
3 kb
1
1
2
2
3
3
4
4
5
5
6
6
1
1
2
2
3
3
4
4
5
5
6
6
1
1
2
2
3
3
4
4
5
5
6
6
1 kb
Andrea
Sarah
Jessica
So you have a positive PCR product:
Now what?
• Clone and sequence clones
• Go straight into sequencing (454)
• Get “community fingerprint” via DGGE and
sequence bands
• Get “community fingerprint” via T-RFLP
Schematic courtesy of B. Crump
The 454 Approach
Schematic courtesy of B. Crump
The 454 Approach
400,000 reads
250 bp in length
Average ~ 250 bp
454
• Avoids laborious/expensive cloning procedures
• Sample diversity deeply and quickly
• Find “rare” or low abundance organisms
• Limited to short reads (<250bp)
• 454 has revolutionized sequencing- many
new technologies every day!
What if we wanted to compare the
general community composition of
each column rather than getting
detailed taxonomic information?
FINGERPRINTING
METHODS
Schematic courtesy of B. Crump
Schematic courtesy of B. Crump
Schematic courtesy of B. Crump
Now we know who is there:
What next?
• Let’s say we have identified an interesting
sequence that we want to know more about in
the column and quantify it
– FISH
– Dot Blot
– Q-PCR
Head et al. 1998
Fluorescent In-Situ Hybridization (FISH)
Schematic courtesy of B. Crump
Fluorescent In-Situ Hybridization (FISH)
Schleper et al. 2005
Schematic courtesy of B. Crump
Schematic courtesy of B. Crump
Quantitative (Real Time) PCR
Real time PCR monitors the fluorescence
emitted during the reactions as an indicator of
amplicon production at each PCR cycle (in
real time) as opposed to the endpoint detection
Quantitative (Real Time) PCR
• Detection of “amplification-associated
fluorescence” at each cycle during PCR
• No gel-based analysis
• Computer-based analysis
• Compare to internal standards
• Must insure specific binding of probes/dye
Quantitative PCR
Some Problems with PCR
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Inhibitors in template DNA
Amplification bias
Gene copy number
Limited by primer design
Differential denaturation efficiency
Chimeric PCR products may form
Contamination w/ non-target DNA
Potentially low sensitivity and resolution
Metagenomics
a.k.a., Community Genomics, Environmental Genomics
Does not rely on Primers or Probes (apriori knowledge)!
Image courtesy of John Heidelberg
Metagenomics
Metagenomics
Metagenomics
Access genomes of uncultured microbes:
Functional Potential
Metabolic Pathways
Horizontal Gene Transfer
…
From the Most “Simple” Microbial
Communities…
•Acid Mine Drainage (pH ~0!)
•Jillian Banfield (UC Berkeley)
•Well-studied, defined environment with ~4
dominant members
•Were able to reconstruct almost entire
community “metagenome”
•Tyson et al. 2004
… to the potentially most diverse!
Venter et al. 2004
•The Sorcerer II Global Ocean Sampling Expedition
•J. Craig Venter Institute “Sequence now, ask questions later”
•Very few genomes reconstructed
•Sequenced 6.3 billion DNA base pairs (Human genome is ~3.2) from top 5 m of ocean
•Discovered more than 6 million genes… and they are only halfway done!
Most of these methods are “who is
there” not “who is active”
• Use RNA
• Link FISH with activity/uptake
DNA
Transcription
mRNA
Translation
Ribosome
Protein
Reverse Transcription PCR (RT-PCR)
• Looks at what genes are being expressed in the environment
• Isolate mRNA
• Reverse transcribe mRNA to produce complementary DNA
(cDNA)
• Amplify cDNA by PCR
• Analzye genes from environment
RT-PCR
• RNA + Reverse Transcriptase + dNTPs= cDNA
• cDNA + Primers + Taq + dNTPs = gene of interest
• Who is active? What genes are active?
Metatranscriptomics
Access expressed genes of uncultured microbes
Schematic courtesy of B. Crump
(Some) Problems with Molecular Methods
DNA extraction
Incomplete sampling
Resistance to cell lysis
Storage
Enzymatic degradation
PCR
Inhibitors in template DNA
Amplification bias
Gene copy number
Fidelity of PCR
Differential denaturation efficiency
Chimeric PCR products
Anytime
Contamination w/ non-target DNA
The “best approach?”
• A little bit of everything!
And the list goes on…
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Optical tweezers
Single cell genomics
Meta-proteomics
Microarrays
Flow Cytometry
Nano-SIMS FISH
In-situ PCR and FISH
…