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Chapter 7 : Stable Isotope Probing Techniques Using N

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Abstract:

This chapter examines technical considerations associated with the use of N in nucleic acid stable isotope probing (SIP) experiments, discusses concerns that should be considered prior to undertaking a N-labeling experiment, and provides an overview of different applications of N-SIP. The preferable approach is to use quantitative PCR (qPCR) to determine the number of 16S rRNA genes in each gradient fraction. The gene target for qPCR analysis can vary by application, and it may be desirable to use universal 16S rRNA gene-targeted primers or primers that are specific to individual domains, individual subgroups, or genera. In DNA purified by secondary gradient fractionation, H genes similar to represented 53% of those recovered while -like sequences represented 17% of those recovered. However, in an experiment in which N-DNA-SIP was used to examine nitrogen-fixing methanotrophs in soil. There are several reasons why N-DNA-SIP represents an appealing method for examining nitrogen (N)-fixing organisms. First, incubations can be carried out at realistic concentrations of substrate, as air can be evacuated from sealed containers and replaced with simulated air containing N. Second, since nitrogen fixation is inhibited in the presence of mineral forms of nitrogen, problems associated with isotope dilution can largely be ignored. Experiments will need to be performed with pure cultures and environmental samples to determine whether N-RNA-SIP can be used effectively in microbial ecology studies.

Citation: Buckley D. 2011. Stable Isotope Probing Techniques Using N, p 129-147. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch7

Key Concept Ranking

Dissimilatory Nitrate Reduction to Ammonia
0.4692913
Restriction Fragment Length Polymorphism
0.4545027
Denaturing Gradient Gel Electrophoresis
0.4545027
Restriction Fragment Length Polymorphism
0.4545027
Denaturing Gradient Gel Electrophoresis
0.4545027
0.4692913
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Figures

Image of FIGURE 1.
FIGURE 1.

Effect of varying the atom% N of NH4Cl on the CsCl buoyant density of DNA from grown in minimal media. Reprinted from , Supplementary Materials) with permission of the publisher.

Citation: Buckley D. 2011. Stable Isotope Probing Techniques Using N, p 129-147. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch7
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Image of FIGURE 2.
FIGURE 2.

Expected relationship between genome G+C content and buoyant density in a CsCl gradient for unlabeled DNA and DNA that is partially or completely labeled with N (see legend). The shaded region of the chart represents the range of densities over which unlabeled DNA would be expected to occur based biologically meaningful values of genome G+C content (30% to 80% G+C content). Reprinted from with permission of the publisher.

Citation: Buckley D. 2011. Stable Isotope Probing Techniques Using N, p 129-147. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch7
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Image of FIGURE 3.
FIGURE 3.

Ability of bis-benzimide to disentangle the effects of stable isotope incorporation and genome G+C content on DNA buoyant density in CsCl gradients. The genome G+C contents of and are approximately 51% and 67%, respectively. (A) In the absence of bis-benzimide, it is impossible to resolve 100% N-labeled DNA from un-labeled DNA. Dashed lines represent unlabeled DNA (○), 100% N-labeled DNA (●), and unlabeled DNA (Δ). The solid line represents a mixture of unlabeled DNA, 100% N-labeled DNA, and unlabeled DNA (□). (B) In the presence of bis-benzimide, it is possible to resolve 100% N-labeled DNA from unlabeled DNA in a mixture of the two (solid line, ●). The density distribution of a pure sample of unlabeled DNA from a secondary gradient containing bis-benzimide is depicted for reference (dashed line, Δ). Reprinted from with permission of the publisher.

Citation: Buckley D. 2011. Stable Isotope Probing Techniques Using N, p 129-147. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch7
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Image of FIGURE 4.
FIGURE 4.

Theoretical relationship between the length of a DNA fragment with a buoyant density of 1.71 g ml and the time required for that DNA fragment to reach equilibrium in a CsCl density gradient of mean density of 1.69 g ml formed in a TlA110 rotor at 55 k rpm (164,000 × ). Reprinted from , Supplementary Materials) with permission of the publisher.

Citation: Buckley D. 2011. Stable Isotope Probing Techniques Using N, p 129-147. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch7
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Image of FIGURE 5.
FIGURE 5.

T-RFLP analysis of 16S rRNA genes can be performed to examine the buoyant density distribution of individual TRFs in primary gradient fractions. Symbols correspond to a TRF of 144 bp length generated by 16S rRNA gene TRFLP analysis of DNA from soil incubated either in artificial air (○) or in artificial air containing N (●). TRF peak height was normalized as a function of the maximum peak height in each gradient. Reprinted from with permission of the publisher.

Citation: Buckley D. 2011. Stable Isotope Probing Techniques Using N, p 129-147. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch7
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Tables

Generic image for table
TABLE 1

Estimated buoyant density of DNA as a function of mol% genome G+C content (G+C) for both unlabeled and 100% N-labeled DNA

Citation: Buckley D. 2011. Stable Isotope Probing Techniques Using N, p 129-147. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch7

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