Molecular Methods in Microbial Ecology
Contact Info: Julie HuberLillie [email protected]
Schedule: 22 Sept: Introductory Lecture, DNA extraction24 Sept: Run DNA products on gel
Lecture on PCR Prepare PCR reactions
29 Sept: 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
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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?
DNA
mRNA
Transcription
The Solution: Molecular Biology
Protein
TranslationRibosome
•Present in all cells- Bacteria, Archaea and Eukaryotes
•Documents of evolutionary history
•Basis of all molecular biological techniques
Head et al. 1998
Head et al. 1998
DNA extraction from Winogradsky Columns
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
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
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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L RB MC GG AS BP LS
Genomic DNA
The sum total of all DNA from an organism or a community of organisms
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
Head et al. 1998
Head et al. 1998
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
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
SSU rRNA
Universal Tree of Life
BACTERIA
EUKARYA
ARCHAEA
Modified from Norman Pace
BACTERIA
EUKARYA
You Are Here
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:95ºC54ºC72ºC
1 minute1 minute1 minute
DNA denaturizationPrimer annealingExtension creation
72ºC 10 minutes Final extension created
Slide courtesy of Byron Crump
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…
Beyond 16S
• Identical 16S = Identical Function
• Target functional genes
Luton et al. 2002
16S rDNA mcrA
Primers we are using• 16S rRNA Bacteria
• 16S rRNA Archaea
• mcrA Methanogens – Methyl coenzyme M reductase
• dsrB Sulfate reducers – Dissimilatory bisulfite reductase
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
Day 3
• Examine gels from DNA and PCR
• Learn about more molecular methods in microbial ecology
Class DNA
Nobu Monica Kenly Marshall
10 kb
3 kb
500 bp
Carrie Chrissy Amy Haruka
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
• General screw-ups
Carrie Marshall Chrissy Kenly
Amy Nobu Haruka Monica
3 kb
500 bp
3 4 2 1
3 4 2 1
3 4 2 1 3 4 2 1 3 4 2 1
3 4 2 1 3 4 2 1 3 4 2 1
3 kb
500 bp
So you have a positive PCR product: Now what?
• Get “community fingerprint” via T-RFLP
• Get “community fingerprint” via DGGE and sequence bands
• Clone and sequence clones
• Go straight into sequencing (massively parallel sequencing, MPS)
B. Crump
B. Crump
B. Crump
What do you DO with sequences?
• Perform a similarity search (database)
• Align the sequences (common ancestry)
• Build a tree (phylogeny and taxonomy)
BLASTBasic Local Alignment Search Tool
http://blast.ncbi.nlm.nih.gov/Blast.cgi
BLASTBasic Local Alignment Search Tool
http://blast.ncbi.nlm.nih.gov/Blast.cgi
Align Sequences and Relatives
Build a Tree (Phylogeny)
Reconstructing evolutionary history and studying the patterns of relationships among organisms
Classification (who is who)
Luton et al. 2002
16S rDNA mcrA
B. Crump
B. Crump
• Built clone libraries from deep-sea rocks
• Compared them to one another and other habitats
Santelli et al. 2008
Santelli et al. 2008
Community Overlap
Santelli et al. 2008
So you have a positive PCR product: Now what?
• Get “community fingerprint” via T-RFLP
• Get “community fingerprint” via DGGE and sequence bands
• Clone and sequence clones
• Go straight into sequencing (massively parallel sequencing, MPS)
Schematic courtesy of B. Crump
MPS Approaches
From Hugenholtz and Tyson 2008
Platform Million base pairs per run
Cost per base (cents)
Average read length (base pairs)
Dye-terminator (ABI 3730xl)
0.07 0.1 700
454-Roche pyrosequencing (GSFLX titanium)
400 0.003 400
Illumina sequencing (GAii)
2,000 0.0007 35
3,000 species?
How many species in 1 L of vent fluid?
3,000 species?
> 36,000 species!
How many species in 1 L of vent fluid?
Now we know who is there:What next?
• Quantify particular groups: FISH or qPCR
Head et al. 1998
Fluorescent In-Situ Hybridization (FISH)
B. Crump
B. Crump
Fluorescent In-Situ Hybridization (FISH)
Schleper et al. 2005
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
• 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
Quantitative PCR
Now we know who and how many:What next?
• Metagenomics
• RNA-based methods
• Many many more…
Metagenomics a.k.a., Community Genomics, Environmental Genomics
Does not rely on Primers or Probes (apriori knowledge)!
Image courtesy of John Heidelberg
Metagenomics
Metagenomics
Access genomes of uncultured microbes:Functional PotentialMetabolic Pathways
Horizontal Gene Transfer…
Metagenomics
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!
•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
Most of these methods are “who is there” not “who is active”
• Use RNA
• Link FISH with activity/uptake
DNA
mRNA
Transcription
Protein
TranslationRibosome
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
• Analyze 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
(Some) Problems with Molecular Methods
D/RNA 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…
• Optical tweezers• Single cell genomics• Meta-proteomics• Microarrays• Flow Cytometry• Nano-SIMS FISH• In-situ PCR and FISH• …
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