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DGGE, T-RFLP, T-RFLP, LH-PCR, ARISA, Melt LH-PCR, Curves

Carli Bober OCN 750 11.03.05

Analyzing the Acronyms

DGGE

Denaturing Gradient Gel Electrophoresis

T-RFLP

Terminal Restriction Fragment Length Polymorphisms

LH-PCR

Length Heterogeneity PCR

ARISA

Automated rRNA Intergenic Spacer Analysis

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Melt Curves

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Melt Curves

· Temperature of a given sample is increased while the change in fluorescence is measured · This will peak at the melting temperature (Tm) · At the melting point, the two strands of DNA will separate and the fluorescence rapidly decreases · The software plots the rate of change of the relative fluorescence units (RFU) with time (T)

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Melt Curves

· Tm= T at which dsDNA melts or denatures · Thermal stability is determined by

­ Base Composition · AT = 2 H bonds · GC = 3 H bonds = Tm due to stacking interactions between neighboring base pairs. ­ DNA fragment length · Shorter = single transition temperature peak Longer (>1000bp) = complex Tm curves due to internal melting domains

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Melt Curves

· Useful QC to check PCR products · All PCR products for a particular primer pair should have the same melting temperature unless there is

­ Contamination ­ Mispriming ­ Primer-dimer artifacts ­ or some other problem

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DGGE

Prepare denaturing gradient gel (formamide and urea)

0% and 80% denaturant

Run PCR product with GC clamp

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DGGE

DNA molecule may have several melting domains with characteristic melting temperatures (Tm) determined by the nucleotide sequence.

The hydrogen bonds formed between complimentary base pairs The attraction between neighbouring bases of the same strand or "stacking"

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DGGE

· Even one nucleotide difference = different Tm's · Mobility of the molecule is retarded at the concentration at which the DNA strands dissociate · The branched structure becomes entangled in the gel matrix and stops moving · Complete strand separation is prevented by a high melting domain, artificially created via a GC clamp

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DGGE

ADVANTAGES High detection rate and sensitivity Methodology is simple and a non-radioactive PCR fragments may be isolated from the gel and used in sequencing reactions DISADVANTAGES Purchase of DGGE equipment may be required Primers are more expensive because of the 40 bases of GC clamp Additional primers may be required for sequencing Genes which are exceptionally GC rich are not easily analyzed by DGGE Analysis of PCR fragments over 400bp is less successful.

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T-RFLP

Terminal restriction fragment length polymorphism (T-RFLP)

Allows the fingerprinting of a community by analyzing the polymorphism of a certain gene

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T-RFLP

Amplify any target gene Cut PCR product with one or more restriction enzymes Laser reader detects the labeled fragments and generates a profile based on fragment lengths

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T-RFLP

High-throughput Reproducible Semi-quantitative analysis of the diversity of a particular gene in a community

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LH-PCR

Based on the natural length variation

within 16S rDNA genes

Amplify small-subunit (SSU) rDNA

Three major variable regions of the SSU can be amplified using different primers Natural variability for one such region varies between 312 and 363 bp

(Suzuki et. al., 1999) 11/3/2005 14

LH-PCR

The variable region is amplified by PCR

with fluorescently labeled universal primers

The peak intensities within each LH-PCR size class are assumed to be proportional to the original template concentrations

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LH-PCR

Advantages

Easy Rapid Reproducible

Disadvantages

Limited by the bacterial species that have been submitted to public databases No exhaustive fragment length database to directly compare and associate LH-PCR lengths with native microorganisms

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ARISA

Automated rRNA Intergenic Spacer Analysis

Amplify Intergenic Spacer (ITS) region between 16S and 23S genes Reported ITS regions vary between 143 and 1,529 bp Use natural variability of ITS region to compare microbial communities among samples

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DNA is pooled and 16S rRNA + intergenic spacer region are amplified by PCR

Collection of Samples Analyzed as a group

ARISA

PCR products cloned to separate amplicons

ARISA profiles represent the bacterial community composition

Size tolerances are generated

Matches allow hypotheses about the species present

Phylogenetic assignment is made for each clone based on a database of known sequences

Clone library is generated

16S RNA gene sequence determined

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ARISA fragment size is determined for each clone, associating a specific 18 sequence with an ARISA fragment size

DISCUSSION

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Crosby and Criddle, 2003

Figure 1

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Crosby and Criddle, 2003

Table 1

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Crosby and Criddle, 2003

Table 2

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Crosby and Criddle, 2003

Figure 2

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Crosby and Criddle, 2003

Figure 3

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West and Scanlan, 1999

Table 1

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West and Scanlan, 1999

Figure 1

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West and Scanlan, 1999

Figure 2

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West and Scanlan, 1999

Figure 3

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West and Scanlan, 1999

Figure 4

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West and Scanlan, 1999

Figure 5

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Tiirola et al., 2002

Table 1

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Tiirola et al., 2002

Table 2

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Tiirola et al., 2002

Figure 1

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Tiirola et al., 2002

Figure 2

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Tiirola et al., 2002

Table 3

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