molecular methods in microbial ecology
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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Molecular Methods in Microbial Ecology
Contact Info: Julie HuberLillie [email protected]
Schedule: 21 October: Introductory Lecture, DNA extraction23 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.
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Day 1
• Introduction to molecular methods in microbial ecology
• Extract DNA from Winogradsky Columns
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Habitat Culturability (%)Seawater 0.001-0.1
Freshwater 0.25Sediments 0.25
Soil 0.3
From Amann et al. 1995 Microbiological Reviews
The Challenge for Microbial Ecology
How do you study something you can’t grow in the lab?
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DNA
mRNA
Transcription
The Solution: Molecular Biology
Protein
TranslationRibosome
•Are present in all cells- Bacteria, Archaea and Eukaryotes
•Are documents of evolutionary history
•Are the basis of all molecular biological techniques
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Head et al. 1998
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Head et al. 1998
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DNA extraction from Winogradsky Columns
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DNA Extraction1. Lyse cell membrane
a. Chemically detergentb. 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
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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
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Head et al. 1998
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Head et al. 1998
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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
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Molecular Biology
DNA
mRNA
Transcription
Protein
TranslationRibosome
•Are present in all cells- Bacteria, Archaea and Eukaryotes
•Are documents of evolutionary history
•Are the basis of all molecular biological techniques
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Ribosomes
• Make proteins
• rRNA is transcribed from rDNA genes
70S Ribosome
50S subunit
30S subunit
21 different proteins
16S rRNA
31 different proteins
23S rRNA 5S rRNA
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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
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SSU rRNA
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Universal Tree of Life
BACTERIA
EUKARYA
ARCHAEA
Modified from Norman Pace
BACTERIA
EUKARYA
You Are Here
ARCHAEA
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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
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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
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Typical PCR Profile
Temperature Time Action
95ºC 5 minutes DNA Taq polymerase activation
35 cycles of:95ºC54ºC72ºC
1 minute1 minute1 minute
DNA denaturizationPrimer annealingExtension creation
72ºC 10 minutes Final extension created
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Slide courtesy of Byron Crump
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Things you can optimize
• 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…
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Primers we are using
• 16S Bacteria
• 16S Archaea
• mcr Methanogens (Methyl coenzyme M reductase)
• dsr Sulfate reducers (Dissimilatory bisulfite reductase
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Reagent Volume (µl) per reaction # of reactions final volume Sterile H20 22.7 5X PCR buffer 10 dNTPs (8mM) 5 Taq polymerase (5 Units/µl) 0.3 Tube Master mix Target Template Vol F primer Vol R primer Vol
µl µl µl 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 + control M. jannaschii
2 20F 5 958R 5
6 38 Nothing - control (water) 2 20F 5 958R 5
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Day 3
• Examine gels from PCR
• Learn about more molecular methods in microbial ecology
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Andrea
Paliza
Jessica
Sarah
Rob
Amy
Class DNA10 kb
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-5
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5
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15
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0 200 400 600 800 1000 1200
Methane Concentration (uM)
Dep
th (c
m)
Col 1
Col 2
Col 4
Col 5
Col 7
Col 8
Rob
Sarah
Paliza
Jessica
Andrea
Amy
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-5
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0 10 20 30 40
Hydrogen Sulfide Concentration (mM)
Dep
th (c
m)
Col 1
Col 2
Col 4
Col 5
Col 7
Col 8
Rob
Sarah
Paliza
Jessica
Andrea
Amy
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1
4
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Rob
Paliza
Amy
Andrea
Sarah
Jessica
3 kb 1 kb 3 kb 1 kb
1
4
3
2
5
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1
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2
5
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1
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1
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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
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Schematic courtesy of B. Crump
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Schematic courtesy of B. Crump
The 454 Approach
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The 454 Approach
400,000 reads 250 bp in length
Average ~ 250 bp
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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!
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What if we wanted to compare the general community composition of
each column rather than getting detailed taxonomic information?
FINGERPRINTING METHODS
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Schematic courtesy of B. Crump
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Schematic courtesy of B. Crump
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Schematic courtesy of B. Crump
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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
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Head et al. 1998
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Fluorescent In-Situ Hybridization (FISH)
Schematic courtesy of B. Crump
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Schematic courtesy of B. Crump
Fluorescent In-Situ Hybridization (FISH)
Schleper et al. 2005
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Schematic courtesy of B. Crump
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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
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• 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 (Real Time) PCR
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Quantitative PCR
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Some Problems with PCR
• 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
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Metagenomics a.k.a., Community Genomics, Environmental Genomics
Does not rely on Primers or Probes (apriori knowledge)!
Image courtesy of John Heidelberg
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Metagenomics
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Metagenomics
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Access genomes of uncultured microbes:Functional PotentialMetabolic Pathways
Horizontal Gene Transfer…
Metagenomics
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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
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… to the potentially most diverse!
•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!
Venter et al. 2004
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Most of these methods are “who is there” not “who is active”
• Use RNA
• Link FISH with activity/uptake
DNA
mRNA
Transcription
Protein
TranslationRibosome
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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
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RT-PCR
• RNA + Reverse Transcriptase + dNTPs= cDNA
• cDNA + Primers + Taq + dNTPs = gene of interest
• Who is active? What genes are active?
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Metatranscriptomics
Access expressed genes of uncultured microbes
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Schematic courtesy of B. Crump
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(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
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The “best approach?”
• A little bit of everything!
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And the list goes on…
• Optical tweezers• Single cell genomics• Meta-proteomics• Microarrays• Flow Cytometry• Nano-SIMS FISH• In-situ PCR and FISH• …