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Chapter 1 : DNA Stable Isotope Probing

Authors: Yin Chen1, J. Colin Murrell2
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Affiliations: 1: School of Life Sciences, University of Warwick, Coventry, CV4 7AL, United Kingdom; 2: School of Life Sciences, University of Warwick, Coventry, CV4 7AL, United Kingdom
Content Type: Monograph
Publication Year: 2011

Category: Environmental Microbiology; Applied and Industrial Microbiology

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Abstract:

The major obstacle for N-based DNA stable isotope probing (DNA-SIP) was insufficient separation of N-labeled and unlabeled DNA due to the lower nitrogen content in DNA compared with its carbon content. Extracted DNA can be loaded onto a cesium chloride (CsCl) gradient for isopycnic centrifugation and separation of labeled “heavy” DNA from unlabeled background DNA (“light” DNA). DNA-SIP experiments need to be implemented carefully in order to maximize achievable information and to avoid misinterpretation of resulting data. Using DNA-SIP, a study demonstrated the possibility that phylum TM7 is involved in toluene degradation in soils. Methanol, formaldehyde, and ammonium are the final products of the RDX degradation pathway. It is obvious that microorganisms that only use nitro-nitrogen in the ring-labeled N-RDX would not be seen in this study. In a study, the authors used DNA-SIP to analyze the active methanotroph population in a peatland from the United Kingdom. By using DNA-SIP, the authors demonstrated that and are probably the most active methane utilizers in this peatland. DNA-SIP can be used in combination with metagenomics in a focused way to investigate the function of a subpopulation of environmental microorganisms. It is predicted that this approach will be adopted by more researchers in the near future. The 16S rRNA gene sequences retrieved from the SIP experiments were used to design specific probes targeting 16S rRNA gene of spp. and spp.

Citation: Chen Y, Murrell J. 2011. DNA Stable Isotope Probing, p 3-24. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch1
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DNA Stable Isotope Probing
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Citation: Chen Y, Murrell J. 2011. DNA Stable Isotope Probing, p 3-24. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch1
/content/10.1128/9781555816896.ch01.ch01fig01
FIGURE 1
The numbers of papers published on stable-isotope probing since the first publication of DNA-SIP in 2000. Search was carried out using the Scopus database using key words “stable isotope probing” or “stable-isotope probing.”
10.1128/9781555816896/f0004-01_thmb.gif
10.1128/9781555816896/f0004-01.gif
Image of FIGURE 1
FIGURE 1

The numbers of papers published on stable-isotope probing since the first publication of DNA-SIP in 2000. Search was carried out using the Scopus database using key words “stable isotope probing” or “stable-isotope probing.”

Citation: Chen Y, Murrell J. 2011. DNA Stable Isotope Probing, p 3-24. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch1
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/content/10.1128/9781555816896.ch01.ch01fig02
FIGURE 2.
An overview of key steps in DNA-SIP.
10.1128/9781555816896/f0011-01_thmb.gif
10.1128/9781555816896/f0011-01.gif
Image of FIGURE 2.
FIGURE 2.

An overview of key steps in DNA-SIP.

Citation: Chen Y, Murrell J. 2011. DNA Stable Isotope Probing, p 3-24. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch1
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/content/10.1128/9781555816896.ch01.ch01fig03
FIGURE 3.
DGGE fingerprints of 16S rRNA genes of key fractions from DNA-SIP. Bands that are highlighted were reamplified and sequenced.
10.1128/9781555816896/f0017-01_thmb.gif
10.1128/9781555816896/f0017-01.gif
Image of FIGURE 3.
FIGURE 3.

DGGE fingerprints of 16S rRNA genes of key fractions from DNA-SIP. Bands that are highlighted were reamplified and sequenced.

Citation: Chen Y, Murrell J. 2011. DNA Stable Isotope Probing, p 3-24. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch1
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/content/10.1128/9781555816896.ch01.ch01fig04
FIGURE 4.
An overview of conventional DNA-SIP, combining DNA-SIP with metagenomics (“focused metagenomics”) and combining DNA-SIP with single cell analysis techniques.
10.1128/9781555816896/f0018-01_thmb.gif
10.1128/9781555816896/f0018-01.gif
Image of FIGURE 4.
FIGURE 4.

An overview of conventional DNA-SIP, combining DNA-SIP with metagenomics (“focused metagenomics”) and combining DNA-SIP with single cell analysis techniques.

Citation: Chen Y, Murrell J. 2011. DNA Stable Isotope Probing, p 3-24. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch1
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Tables

DNA Stable Isotope Probing
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Citation: Chen Y, Murrell J. 2011. DNA Stable Isotope Probing, p 3-24. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch1
/content/10.1128/9781555816896.ch01.ch01tab01
TABLE 1
Key studies using DNA-SIP for identifying active microorganisms from diverse habitats
Generic image for table
TABLE 1

Key studies using DNA-SIP for identifying active microorganisms from diverse habitats

Citation: Chen Y, Murrell J. 2011. DNA Stable Isotope Probing, p 3-24. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch1
/content/10.1128/9781555816896.ch01.ch01tab02
TABLE 2
Comparison of DNA-, RNA-, PLFA- and Protein-SIP
Generic image for table
TABLE 2

Comparison of DNA-, RNA-, PLFA- and Protein-SIP

Citation: Chen Y, Murrell J. 2011. DNA Stable Isotope Probing, p 3-24. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch1

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