Molecular Methods in Microbial Ecology

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Molecular Methods in Microbial Ecology

• Contact Info Julie Huber
• Lillie 305
• x7291
• jhuber_at_mbl.edu
• Schedule 22 Sept Introductory Lecture, DNA
  extraction
• 24 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.

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Day 1

• Introduction to molecular methods in microbial
  ecology
• Extract DNA from Winogradsky Columns

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The Challenge for Microbial Ecology
Habitat Culturability ()
Seawater 0.001-0.1
Freshwater 0.25
Sediments 0.25
Soil 0.3
How do you study something you cant grow in the
lab?
From Amann et al. 1995 Microbiological Reviews
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The Solution Molecular Biology
DNA
Transcription
mRNA
Translation
Ribosome
Protein

• Present in all cells- Bacteria, Archaea and
  Eukaryotes
• Documents of evolutionary history
• 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 Extraction

• Lyse cell membrane
• Chemically ? detergent
• Physically ? bead beating
• Pellet cell membrane, proteins and other cell
  parts while DNA stays in solution
• Remove other inhibitors from DNA
• Mix DNA with acid and salt ? stick to filter
• Wash filter-bound DNA several times with alcohol
• 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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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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Genomic DNA The sum total of all DNA from an
organism or a community of organisms
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What to do

• Mix 10 µl of your DNA with 2 µl loading buffer
• Load in well on gel
• Ill load the ladder
• Run it
• Take a picture of it

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Head et al. 1998
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Head et al. 1998
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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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Ribosomes

• Make proteins
• rRNA is transcribed from rDNA genes

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SSU rRNA
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Universal Tree of Life
BACTERIA
BACTERIA
ARCHAEA
ARCHAEA
You Are Here
EUKARYA
EUKARYA
Modified from Norman Pace
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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 of95ºC54ºC72ºC 1 minute 1 minute 1 minute DNA denaturization Primer annealing Extension 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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Beyond 16S

• Identical 16S Identical Function
• Target functional genes

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16S rDNA
mcrA
Luton et al. 2002
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Primers we are using

• 16S rRNA Bacteria
• 16S rRNA Archaea
• mcrA Methanogens
• Methyl coenzyme M reductase
• dsrB Sulfate reducers
• Dissimilatory bisulfite reductase

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Day 3

• Examine gels from DNA and PCR
• Learn about more molecular methods in microbial
  ecology

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Class DNA
Nobu Monica Kenly Marshall
Carrie Chrissy Amy Haruka
10 kb
3 kb
500 bp
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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
• General screw-ups

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Amy Nobu Haruka Monica
3 kb
500 bp
3 4 2 1
3 4 2 1
3 4 2 1
3 4 2 1
Carrie Marshall Chrissy Kenly
3 kb
500 bp
3 4 2 1
3 4 2 1
3 4 2 1
3 4 2 1
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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)

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B. Crump
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B. Crump
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B. Crump
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What do you DO with sequences?

• Perform a similarity search (database)
• Align the sequences (common ancestry)
• Build a tree (phylogeny and taxonomy)

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BLASTBasic Local Alignment Search Tool
http//blast.ncbi.nlm.nih.gov/Blast.cgi
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BLASTBasic Local Alignment Search Tool
http//blast.ncbi.nlm.nih.gov/Blast.cgi
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Align Sequences and Relatives
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Build a Tree (Phylogeny)

• Reconstructing evolutionary history and studying
  the patterns of relationships among organisms

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Classification (who is who)
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16S rDNA
mcrA
Luton et al. 2002
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B. Crump
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B. Crump
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• Built clone libraries from deep-sea rocks
• Compared them to one another and other habitats

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Santelli et al. 2008
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Santelli et al. 2008
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Community Overlap
Santelli et al. 2008
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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)

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MPS Approaches
Schematic courtesy of B. Crump
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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
From Hugenholtz and Tyson 2008
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How many species in 1 L of vent fluid?
3,000 species?
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How many species in 1 L of vent fluid?
gt 36,000 species!
3,000 species?
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Now we know who is thereWhat next?

• Quantify particular groups FISH or qPCR

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Head et al. 1998
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Fluorescent In-Situ Hybridization (FISH)
B. Crump
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Fluorescent In-Situ Hybridization (FISH)
Schleper et al. 2005
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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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

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Quantitative PCR
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Now we know who and how manyWhat next?

• Metagenomics
• RNA-based methods
• Many many more

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Metagenomics a.k.a., Community Genomics,
Environmental GenomicsDoes 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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Metagenomics
Access genomes of uncultured microbes Functional
Potential Metabolic Pathways Horizontal Gene
Transfer
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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!
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!

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Most of these methods are who is there not who
is active

• Use RNA
• Link FISH with activity/uptake

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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
• Analyze 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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(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
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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