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Chapter 55 : PCR: Agricultural and Environmental Applications for Soil Microbes

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Abstract:

This chapter introduces the primary concepts behind PCR, describes different flavors or specialized derivatives of PCR that are used to overcome various problems, provides an overview of ways in which the use of PCR can enhance soil microbiology research, and also provides information on equipment and other products designed for PCR. Ultimately, the PCR copies and amplifies the target sequence from the nucleic acid template, doubling the number of copies during each cycle of temperature. The different PCR methodologies are Nested and Seminested PCRs, Touchdown PCR, hot-start PCR, Booster PCR, Two-Step PCR, Multiplex PCR and Reverse transcriptase (RT) PCR. In recent years there has been increased interest in the use of DNA fingerprinting methodologies to identify bacteria at the isolate level and to subtype pathogenic bacteria. A more recent method, terminal-restriction fragment length polymorphism (T-RFLP) analysis, has also been used to study microbial community structures. T-RFLP analysis is based on the restriction endonuclease digestion of PCR products that have been fluorescently end labeled through the use of labeled primers. It is probably already apparent that the choice of the primer sequences is critical for the successful amplification of a specific DNA sequence. The degree of specificity can be varied by primer design and also by changing the annealing temperature. Sensitivity can be evaluated in terms of whole-cell lysates or pure genomic DNA preparations. Overall, PCR adds a useful new technology to aid in the study of plant and soil microbes.

Citation: Dowd S, Pepper I. 2007. PCR: Agricultural and Environmental Applications for Soil Microbes, p 676-686. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch55

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Reverse Transcriptase PCR
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Denaturing Gradient Gel Electrophoresis
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Citation: Dowd S, Pepper I. 2007. PCR: Agricultural and Environmental Applications for Soil Microbes, p 676-686. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch55
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Citation: Dowd S, Pepper I. 2007. PCR: Agricultural and Environmental Applications for Soil Microbes, p 676-686. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch55
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Citation: Dowd S, Pepper I. 2007. PCR: Agricultural and Environmental Applications for Soil Microbes, p 676-686. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch55
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Citation: Dowd S, Pepper I. 2007. PCR: Agricultural and Environmental Applications for Soil Microbes, p 676-686. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch55
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Citation: Dowd S, Pepper I. 2007. PCR: Agricultural and Environmental Applications for Soil Microbes, p 676-686. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch55
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References

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Tables

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TABLE 1

Standard PCR mixture

Citation: Dowd S, Pepper I. 2007. PCR: Agricultural and Environmental Applications for Soil Microbes, p 676-686. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch55
Generic image for table
TABLE 2

Companies that manufacture and sell thermocyclers

Citation: Dowd S, Pepper I. 2007. PCR: Agricultural and Environmental Applications for Soil Microbes, p 676-686. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch55
Generic image for table
TABLE 3

Examples of PCR master mixes

Citation: Dowd S, Pepper I. 2007. PCR: Agricultural and Environmental Applications for Soil Microbes, p 676-686. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch55
Generic image for table
TABLE 4

Examples of Q-PCR machines

Citation: Dowd S, Pepper I. 2007. PCR: Agricultural and Environmental Applications for Soil Microbes, p 676-686. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch55
Generic image for table
TABLE 5

Q-PCR reagent kits

Citation: Dowd S, Pepper I. 2007. PCR: Agricultural and Environmental Applications for Soil Microbes, p 676-686. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch55
Generic image for table
TABLE 6

Examples of primer design software

Citation: Dowd S, Pepper I. 2007. PCR: Agricultural and Environmental Applications for Soil Microbes, p 676-686. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch55
Generic image for table
TABLE 7

Examples of PCR optimization kits containing adjuncts and optimization buffers

Citation: Dowd S, Pepper I. 2007. PCR: Agricultural and Environmental Applications for Soil Microbes, p 676-686. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch55
Generic image for table
TABLE 8

Soil DNA extraction kits

Citation: Dowd S, Pepper I. 2007. PCR: Agricultural and Environmental Applications for Soil Microbes, p 676-686. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch55

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