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 Biology10 Oct 2011

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 acidsRNA: ribonucleic acids

Genetic material sequenced after amplification using PCR (polymerase chain reaction) or used in finger-printing approaches

One study found that 6,000-10,000 unique genomes found in soil, compared to 40 cultured organisms

GREAT PLATE ANOMALY

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 REV PRIMERTTAACGGGGCCCTTTAAA AAACCCGGGTTTAAA

The reverse primer is the reverse complement of the end of the gene sequence

Taq Polymerase

Isolated from a THERMOPHILIC bacterium in 1965This allows high temperatures for DNA denaturation$2.3 billion in royaltiesNobel prize

Does introduce errors 1 in ~10,000 bases

Traditional ApproachesAbundance-ID PLFA-Richness

ID of those that can be cultured Bacterial richnessCertain FA identify certain

organisms

Traditional ApproachesMicrobial 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 biomassFC is the CO2 produced by fumigated soilUfC is the CO2 produced by the nonfumigated soil sampleKC is the fraction of microbial biomass C mineralized to CO2 *( a constant of 40-45%)

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)

Taxonomy / Function

Microarrays RFLP

Function – Genes turned on or offNo direct taxonomy yet>15,000 genes on a chipUse soil DNA (presence) or reverse-transcribed RNA (expression)Dependent on sequences in database

Largely outdatedCheapRough scale taxonomy16S rRNA gene

Nucleic Acid Analysis

DNA sequencingCloning

DNA sequencingCloning

Limitations of Cloning:

ExpensiveTime consuming

PCR your gene of interestPurify your gene of interestClone your geneLet colonies growPick coloniesRe-grow picked coloniesSubmit for sequencingTotal time ~ 1 weekCost ~ $1,000 for 96 sequences not including labor (~$14/read)

DNA sequencingHigh Throughput Sequencing - Pyrosequencing

DNA sequencingHigh 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 problematicErrors --- how to deal with that in taxonomyMany steps that are prone to error

DNA sequencingIlumina Sequencing

DNA sequencingHigh 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 problematicErrors --- how to deal with that in taxonomyMany 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 doMt: Collection of all mRNAs in a sample: Who and what they are doing

Gene QuantificationQuantitative PCR (q-PCR)

Quantitative Reverse Transcription PCR (RT-PCR)

Community FingerprintingT-RFLPDGGE

Increase in fluorescence over time

Standard curve of known gene copies

DNA: How many are there?

RNA: What is the real expression of the gene?

Used less often now

T-RFLP like RFLP one end labeled w/dye peaks are size of population

Denaturing gradient gel electrophoresis Cut DNA Melts at different conditions Get a gel pattern Changes in diversity

q-PCR

QUANTITATIVE PCRAbility to count the number of target genesUses a dye that binds to dsDNASame as PCRCamera records increase in fluorescence due to dye binding over time

Can targetDNARNA that is reverse-transcribed

Reverse transcriptionTake ssRNA and convert to dsDNAWhy?

RNA is degraded rapidlyGives 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, PLFATaxonomy/Function: FISH, STARFISH, SIPNucleic Acid Analysis: Microarrays, RFLP, Sequencing

Metagenomics vs. MetatranscriptomicsCloning vs. New Methods

Quantification: qPCR

Broad understanding: Not details!---except for PCRWhich 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)