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DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T DGGE, T - - RFLP, RFLP, LH LH - - PCR, ARISA, Melt PCR, ARISA, Melt Curves Curves Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

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Page 1: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

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

Page 2: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 2

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

Page 3: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 3

Melt CurvesMelt Curves

Page 4: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 4

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)

Page 5: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 5

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

Page 6: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 6

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 ☺

Page 7: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 7

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

Page 8: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 8

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"

Page 9: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 9

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

Page 10: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 10

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.

Page 11: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 11

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

Page 12: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 12

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

Page 13: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 13

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

Page 14: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 14

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)

Page 15: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 15

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

Page 16: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 16

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

Page 17: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

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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

Page 18: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 18

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

Page 19: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 19

DISCUSSIONDISCUSSION

Page 20: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 20

Crosby and Criddle, 2003Crosby and Criddle, 2003Figure 1Figure 1

Page 21: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 21

Crosby and Criddle, 2003Crosby and Criddle, 2003

Table 1Table 1

Page 22: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 22

Crosby and Criddle, 2003Crosby and Criddle, 2003Table 2Table 2

Page 23: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 23

Crosby and Criddle, 2003Crosby and

Criddle, 2003

Figure 2Figure 2

Page 24: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 24

Crosby and Criddle, 2003Crosby and Criddle, 2003Figure 3Figure 3

Page 25: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 25

West and Scanlan, 1999West and Scanlan, 1999

Table 1Table 1

Page 26: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 26

West and Scanlan, 1999

West and Scanlan, 1999

Figure 1Figure 1

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11/3/2005 27

West and Scanlan, 1999West and Scanlan, 1999

Figure 2Figure 2

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11/3/2005 28

West and Scanlan, 1999West and Scanlan, 1999

Figure 3Figure 3

Page 29: DGGE, T-RFLP, LH-PCR, ARISA, Melt Curves - · PDF fileDGGE, T-RFLP, LH-PCR, ARISA, Melt Curves DGGE, T-RFLP, LH-PCR, ARISA, Melt Carli Bober OCN 750 11.03.05 Carli Bober OCN 750 11.03.05

11/3/2005 29

West and Scanlan, 1999

West and Scanlan, 1999

Figure 4Figure 4

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11/3/2005 30

West and Scanlan, 1999West and Scanlan, 1999

Figure 5Figure 5

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11/3/2005 31

Tiirola et al., 2002Tiirola et al., 2002

Table 1Table 1

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11/3/2005 32

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