Post on 06-Feb-2018
DGGE, T-RFLP, LH-PCR, ARISA, Melt
Curves
DGGE, TDGGE, T--RFLP, RFLP, LHLH--PCR, ARISA, Melt PCR, ARISA, Melt
CurvesCurves
Carli BoberOCN 75011.03.05
Carli BoberOCN 75011.03.05
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Analyzing the AcronymsAnalyzing the Acronyms
DGGEDenaturing Gradient Gel Electrophoresis
T-RFLPTerminal Restriction Fragment Length Polymorphisms
LH-PCRLength Heterogeneity PCR
ARISAAutomated rRNA Intergenic Spacer Analysis
DGGEDenaturing Gradient Gel Electrophoresis
T-RFLPTerminal Restriction Fragment Length Polymorphisms
LH-PCRLength Heterogeneity PCR
ARISAAutomated rRNA Intergenic Spacer Analysis
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Melt CurvesMelt Curves
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Melt CurvesMelt 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)
• 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 CurvesMelt 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 peakLonger (>1000bp) = complex Tm curves due
to internal melting domains
• 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 peakLonger (>1000bp) = complex Tm curves due
to internal melting domains
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Melt CurvesMelt 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 ☺
• 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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DGGEDGGE
Prepare denaturing gradient gel (formamide and urea)
0% and 80% denaturant
Run PCR product with GC clamp
Prepare denaturing gradient gel (formamide and urea)
0% and 80% denaturant
Run PCR product with GC clamp
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DGGEDGGEDNA molecule may have several melting domains with characteristic melting temperatures (Tm) determined by the nucleotide sequence.
The hydrogen bonds formed between complimentary base pairsThe attraction between neighbouring bases of the same strand or "stacking"
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DGGEDGGE• 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
• 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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DGGEDGGEADVANTAGES
High detection rate and sensitivityMethodology is simple and a non-radioactivePCR fragments may be isolated from the gel and used in sequencing reactions
DISADVANTAGESPurchase of DGGE equipment may be requiredPrimers are more expensive because of the 40 bases of GC clampAdditional primers may be required for sequencingGenes which are exceptionally GC rich are not easily analyzed by DGGEAnalysis of PCR fragments over 400bp is less successful.
ADVANTAGES High detection rate and sensitivityMethodology is simple and a non-radioactivePCR fragments may be isolated from the gel and used in sequencing reactions
DISADVANTAGESPurchase of DGGE equipment may be requiredPrimers are more expensive because of the 40 bases of GC clampAdditional primers may be required for sequencingGenes which are exceptionally GC rich are not easily analyzed by DGGEAnalysis of PCR fragments over 400bp is less successful.
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T-RFLPT-RFLP
Terminal restriction fragment length polymorphism (T-RFLP)
Allows the fingerprinting of a community by analyzing the polymorphism of a certain gene
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-RFLPT-RFLPAmplify anyAmplify any
target genetarget geneCut PCR product Cut PCR product
with one or more with one or more restriction enzymesrestriction enzymes
Laser reader Laser reader detects the labeled detects the labeled fragments and fragments and generates a profile generates a profile based on fragment based on fragment lengthslengths
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T-RFLPT-RFLP
High-throughputReproducibleSemi-quantitative analysis of the diversity of a particular gene in a community
High-throughputReproducibleSemi-quantitative analysis of the diversity of a particular gene in a community
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LH-PCRLH-PCR
Based on the natural length variation within 16S rDNA genesAmplify small-subunit (SSU) rDNA
Three major variable regions of the SSU can be amplified using different primersNatural variability for one such region varies between 312 and 363 bp
Based on the natural length variation within 16S rDNA genesAmplify small-subunit (SSU) rDNA
Three major variable regions of the SSU can be amplified using different primersNatural variability for one such region varies between 312 and 363 bp
(Suzuki et. al., 1999)
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LH-PCRLH-PCR
The variable region is amplified by PCR with fluorescently labeled universal primersThe peak intensities within each LH-PCR size class are assumed to be proportional to the original template concentrations
The variable region is amplified by PCR with fluorescently labeled universal primersThe peak intensities within each LH-PCR size class are assumed to be proportional to the original template concentrations
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LH-PCRLH-PCR
AdvantagesEasy RapidReproducible
DisadvantagesLimited by the bacterial species that have been submitted to public databasesNo exhaustive fragment length database to directly compare and associate LH-PCR lengths with native microorganisms
AdvantagesEasy RapidReproducible
DisadvantagesLimited by the bacterial species that have been submitted to public databasesNo exhaustive fragment length database to directly compare and associate LH-PCR lengths with native microorganisms
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ARISAARISA
Automated rRNA Intergenic Spacer Analysis
Amplify Intergenic Spacer (ITS) region between 16S and 23S genesReported ITS regions vary between 143 and 1,529 bpUse natural variability of ITS region to compare microbial communities among samples
Automated rRNA Intergenic Spacer Analysis
Amplify Intergenic Spacer (ITS) region between 16S and 23S genesReported ITS regions vary between 143 and 1,529 bpUse natural variability of ITS region to compare microbial communities among samples
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ARISAARISACollection of SamplesAnalyzed as a group ARISA profiles represent the
bacterial community composition
DNA is pooled and 16S rRNA + intergenic spacer region are amplified by PCR
PCR productscloned to separate amplicons
Clone library is generated
ARISA fragment size is determined for each clone, associating a specific sequence with an ARISA fragment size
16S RNA gene sequence
determined
Phylogenetic assignment is made for each clone based on a database of known sequences
Size tolerances are generated
Matches allow hypotheses about
the species present
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DISCUSSIONDISCUSSION
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Crosby and Criddle, 2003Crosby and Criddle, 2003Figure 1Figure 1
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Crosby and Criddle, 2003Crosby and Criddle, 2003
Table 1Table 1
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Crosby and Criddle, 2003Crosby and Criddle, 2003Table 2Table 2
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Crosby and Criddle, 2003Crosby and
Criddle, 2003
Figure 2Figure 2
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Crosby and Criddle, 2003Crosby and Criddle, 2003Figure 3Figure 3
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West and Scanlan, 1999West and Scanlan, 1999
Table 1Table 1
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West and Scanlan, 1999
West and Scanlan, 1999
Figure 1Figure 1
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West and Scanlan, 1999West and Scanlan, 1999
Figure 2Figure 2
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West and Scanlan, 1999West and Scanlan, 1999
Figure 3Figure 3
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West and Scanlan, 1999
West and Scanlan, 1999
Figure 4Figure 4
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West and Scanlan, 1999West and Scanlan, 1999
Figure 5Figure 5
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Tiirola et al., 2002Tiirola et al., 2002
Table 1Table 1
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Tiirola et al., 2002Tiirola et al., 2002
Table 2Table 2
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Tiirola et al., 2002Tiirola et al., 2002
Figure 1Figure 1
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Tiirola et al., 2002Tiirola et al., 2002
Figure 2Figure 2
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Tiirola et al., 2002Tiirola et al., 2002
Table 3Table 3