Transcript Lecture 7 Oct 10th

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76% passed, average=75%, stdev=20%; High score =101%, Low score = 27%
Test was worth 13 points of a total of 100 for the class. Multiply your score by 0.13 for your points.
Methods For Studying Microbial Communities
CSS 360 Soil Biology
10 Oct 2011
GREAT PLATE ANOMALY
Molecular methods to characterize the soil microbial
community have given us insight into the previously
unimagined diversity of soil organisms. These methods
alone, however, don’t tell us about the function of the
soil micro-organisms and should be carried out in
conjunction with methods that do.
DNA: deoxyribonucleic acids
RNA: ribonucleic acids
Genetic material sequenced after amplification using
PCR (polymerase chain reaction) or used in fingerprinting approaches
One study found that 6,000-10,000 unique genomes found in soil,
compared to 40 cultured organisms
PCR-Based Assays
• Technique which is used to amplify the number of copies of a
specific region of DNA, in order to produce enough DNA to be
adequately tested.
• In order to use PCR, one must already know the exact sequences
which flank (lie on either side of) both ends of a given region of
interest in DNA (may be a gene or any sequence). One need not
know the DNA sequence in-between.
PCR-Based Assays
TTAACGGGGCCCTTTAAA................................TTTAAACCCGGGTTT
Target sequence
If these sequences flank (are on either side) of a particular region of a particular
organism's DNA, and NO OTHER ORGANISM'S DNA (or a different size
product). This region would be a target sequence for PCR.
The first step for PCR would be to synthesize "primers" that will be exactly the
same as the flanking sequences shown above. We make ONE primer exactly like
the forward sequence, this is our forward primer, and we make the reverse the
reverse complement of the end sequence
TTAACGGGGCCCTTTAAA........TTTAAACCCGGGTTT
FWD PRIMER
TTAACGGGGCCCTTTAAA
REV PRIMER
AAACCCGGGTTTAAA
The reverse primer is the reverse complement of the end of the gene sequence
Taq Polymerase
Isolated from a THERMOPHILIC bacterium in 1965
This allows high temperatures for DNA denaturation
$2.3 billion in royalties
Nobel prize
Does introduce errors
1 in ~10,000 bases
Abundance-ID
Traditional Approaches
PLFA-Richness
ID of those that can be cultured
organisms
Bacterial richness
Certain FA identify certain
Traditional Approaches
Microbial Biomass: Chloroform Fumigation Incubation (CFI) and
Extraction Methods
CFI: exposes soil to ethanol-free chloroform for 24 h to kill indigenous microbes
Chloroform is removed and the flush of mineralized CO2 and NH4+ are measured
during a 10 d incubation.
What causes this flush? The use of cell lysates as a C and energy source by
organisms that have survived the fumigation (spores or cysts)
Biomass C= (FC-UfC)/KC
Biomass C= amount of C trapped in the microbial biomass
FC is the CO2 produced by fumigated soil
UfC is the CO2 produced by the nonfumigated soil sample
KC is the fraction of microbial biomass C mineralized to CO2 *( a
constant of 40-45%)
Taxonomy / Function
Fluorescence In-Situ Hybridization
STARFISH
Tagged DNA probe complementary to
target sequence. ID or function
Microautoradiography with whole cell
FISH. Radioactive compounds such as
acetate, ammonium, glucose, etc.
ID AND function
Taxonomy / Function
Stable Isotope Probing (SIP)
Nucleic Acid Analysis
Microarrays
Function – Genes turned on or off
No direct taxonomy yet
>15,000 genes on a chip
Use soil DNA (presence) or
reverse-transcribed RNA (expression)
Dependent on sequences in database
RFLP
Largely outdated
Cheap
Rough scale taxonomy
16S rRNA gene
DNA sequencing
Cloning
DNA sequencing
Cloning
Limitations of Cloning:
Expensive
Time consuming
PCR your gene of interest
Purify your gene of interest
Clone your gene
Let colonies grow
Pick colonies
Re-grow picked colonies
Submit for sequencing
Total time ~ 1 week
Cost ~ $1,000 for 96 sequences not including labor (~$14/read)
DNA sequencing
High Throughput Sequencing - Pyrosequencing
DNA sequencing
High Throughput Sequencing - Pyrosequencing
Pyrosequencing (aka 454)
Advantages:
Lower cost (~$6,000 per 800,000 sequences) (1.3 reads/ 1 cent)
Good read length (~600 bases)
Disadvantages:
PCR can be problematic
Errors --- how to deal with that in taxonomy
Many steps that are prone to error
DNA sequencing
Ilumina Sequencing
DNA sequencing
High Throughput Sequencing - Illumina
Pyrosequencing (aka 454)
Advantages:
Lower cost (~$12,000 per 160 million sequences) (133 reads /1 cent)
Good read length (~600 bases)
Disadvantages:
PCR can be problematic
Errors --- how to deal with that in taxonomy
Many steps that are prone to error
Next-Next Generation Sequencing
Metagenomics and Metatranscriptomics
Mg: Collection of all genes in a sample: Who and what they can do
Mt: Collection of all mRNAs in a sample: Who and what they are doing
Quantification
Quantitative PCR (q-PCR)
Quantitative Reverse
Transcription PCR (RT-PCR)
Community Fingerprinting
T-RFLP
DGGE
Used less often now
Increase in fluorescence over time
Standard curve of known gene copies
T-RFLP like RFLP
one end labeled w/dye
peaks are size of population
DNA: How many are there?
RNA: What is the real expression of
the gene?
Denaturing gradient gel
electrophoresis
Cut DNA
Melts at different conditions
Get a gel pattern
Changes in diversity
q-PCR
QUANTITATIVE PCR
Ability to count the number of target genes
Uses a dye that binds to dsDNA
Same as PCR
Camera records increase in fluorescence due to dye binding over time
Can target
DNA
RNA that is reverse-transcribed
Reverse transcription
Take ssRNA and convert to dsDNA
Why?
RNA is degraded rapidly
Gives us the ACTIVITY of the gene, not just presence
Relate these genes to soil processes, such as N or C cycling
KNOW:
Process of PCR, why is it used?
Classical methods: Chloroform, Plating, PLFA
Taxonomy/Function: FISH, STARFISH, SIP
Nucleic Acid Analysis: Microarrays, RFLP, Sequencing
Metagenomics vs. Metatranscriptomics
Cloning vs. New Methods
Quantification: qPCR
Broad understanding: Not details!---except for PCR
Which method would be used for……
Difference between two methods…..in terms of question answered
Knowing more about a method….good bonus question.
• Reading Assignment
• Bardgett 64-69: Phosphorus Cycling
• Bardgett Chapter 4 pp. 86-107(next two
lectures)