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Chapter 41 : Analysis of Microbial Communities with Denaturing Gradient Gel Electrophoresis and Terminal Restriction Fragment Length Polymorphism

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Analysis of Microbial Communities with Denaturing Gradient Gel Electrophoresis and Terminal Restriction Fragment Length Polymorphism, Page 1 of 2

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Abstract:

Microbial communities can exhibit an enormous range of complexity, from those with a mere handful of populations to those with thousands of species derived from all three domains of life. For community analysis, genetic fingerprinting techniques allow the comparative profiling of many environmental samples and thus facilitate the spatial and temporal analysis of microbial communities in ecosystems. Two of these approaches, denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP), are described herein. In reality, complex microbial communities with population richness exceeding several hundred species are beyond single-primer pair PCR analysis. Many would consider the current analytical methods more exploratory in nature, just the beginning to quantitatively address the complex relationships between microbial communities. The marker should be composed of DGGE PCR products, and under no circumstances should size markers be used. T-RFLP has been used as an effective tool in the dissection of microbial communities. The digestions were carried out assuming that PCR products were generated with a labeled 63F bacterial domain-specific primer. Hence, all fragments are derived from the 5'- terminus of bacterial rRNA genes.

Citation: Nakatsu C, Marsh T. 2007. Analysis of Microbial Communities with Denaturing Gradient Gel Electrophoresis and Terminal Restriction Fragment Length Polymorphism, p 909-923. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch41

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Denaturing Gradient Gel Electrophoresis
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Restriction Fragment Length Polymorphism
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Figures

Image of FIGURE 1
FIGURE 1

Flow diagram of the steps for microbial community analysis using either DGGE or T-RFLP.

Citation: Nakatsu C, Marsh T. 2007. Analysis of Microbial Communities with Denaturing Gradient Gel Electrophoresis and Terminal Restriction Fragment Length Polymorphism, p 909-923. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch41
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Image of FIGURE 2
FIGURE 2

Diagram of a melting profile of a single PCR product separated using perpendicular DGGE. The approximate denaturing gradient to use would be 25 to 50% denaturant.

Citation: Nakatsu C, Marsh T. 2007. Analysis of Microbial Communities with Denaturing Gradient Gel Electrophoresis and Terminal Restriction Fragment Length Polymorphism, p 909-923. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch41
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Image of FIGURE 3
FIGURE 3

Diagram of the separation of a two-member community inoculated over 6.6 h at 20-min intervals using parallel DGGE. This example uses a 25 to 60% denaturing gradient and electrophoresis at 200 V and 60°C. The optimal separation time would be 4.5 to 5 h.

Citation: Nakatsu C, Marsh T. 2007. Analysis of Microbial Communities with Denaturing Gradient Gel Electrophoresis and Terminal Restriction Fragment Length Polymorphism, p 909-923. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch41
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Image of FIGURE 4
FIGURE 4

DGGE profiles of PCR products of 16S rDNA communities from bioreactors (lanes 1), lakes (lanes 2), corn rhizosphere (lanes 3), bulk agricultural soils (lanes 4), and soils contaminated with heavy metals and organic solvents (lanes 5). With the exception of the bulk agricultural soils, intense bands are observed in each profile. Shown is an inverted image of DGGE gels stained with SYBR Green I.

Citation: Nakatsu C, Marsh T. 2007. Analysis of Microbial Communities with Denaturing Gradient Gel Electrophoresis and Terminal Restriction Fragment Length Polymorphism, p 909-923. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch41
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Image of FIGURE 5
FIGURE 5

Terminal fragment size distribution. An rRNA database of 20,070 sequences was digested in silico with HaeIII (upper panel) and RsaI (lower panel). The frequency (ordinate) of terminal fragment sizes (abscissa) appearing in the database is presented.

Citation: Nakatsu C, Marsh T. 2007. Analysis of Microbial Communities with Denaturing Gradient Gel Electrophoresis and Terminal Restriction Fragment Length Polymorphism, p 909-923. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch41
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Image of FIGURE 6
FIGURE 6

Number of unique fragments generated with commonly used restriction endonucleases. A 20,070-sequence rRNA database was digested in silico with 19 restriction enzymes (abscissa). The number of unique terminal fragment sizes is reported for each enzyme (ordinate).

Citation: Nakatsu C, Marsh T. 2007. Analysis of Microbial Communities with Denaturing Gradient Gel Electrophoresis and Terminal Restriction Fragment Length Polymorphism, p 909-923. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch41
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Image of FIGURE 7
FIGURE 7

T-RFLP profile of a soil community. Samples were amplified with HEX-labeled 27F primer matched with unlabeled 1492R targeting the 16S rRNA genes. PCR products were digested with HhaI and run on an ABI 273A gel system. T-RFLP profiles were viewed with GeneScan. (A) A 50- to 600-base scale on abscissa; (B) expanded base scale of data from panel A.

Citation: Nakatsu C, Marsh T. 2007. Analysis of Microbial Communities with Denaturing Gradient Gel Electrophoresis and Terminal Restriction Fragment Length Polymorphism, p 909-923. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch41
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Tables

Generic image for table
TABLE 1

Examples of PCR primers for DGGE and T-RFLP

For prokaryotes relative to 16S rRNA gene sequence of and for eukaryotes 18S rRNA gene sequence of

A GC clamp must be added to the 5′ end of one of the primer pairs used for PCR.

GC clamp used 5′ CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G 3′.

GC clamp used 5′ CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC C 3′.

Citation: Nakatsu C, Marsh T. 2007. Analysis of Microbial Communities with Denaturing Gradient Gel Electrophoresis and Terminal Restriction Fragment Length Polymorphism, p 909-923. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch41
Generic image for table
TABLE 2

Standard PCR conditions

Citation: Nakatsu C, Marsh T. 2007. Analysis of Microbial Communities with Denaturing Gradient Gel Electrophoresis and Terminal Restriction Fragment Length Polymorphism, p 909-923. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch41
Generic image for table
TABLE 3

Number of 5′ proximal restriction target sequences on 16S rDNA

Based on an RDP library of 27,060 sequences, each of which is at least 1,200 bases long (REF). The database was screened with PatScan (REF) for primer and restriction sites.

A total of 18,932 out of 27,060 sequences were recognized by the 63F primer when the criteria required a perfect match in the four bases at the 3′ terminus and allowed up to three mismatches in the 5′ proximal 17 bases.

Citation: Nakatsu C, Marsh T. 2007. Analysis of Microbial Communities with Denaturing Gradient Gel Electrophoresis and Terminal Restriction Fragment Length Polymorphism, p 909-923. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch41

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