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Chapter 16 : Microwell Plate Detection Systems for Amplicon Detection and Characterization

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Abstract:

Sequence-specific methods for the detection of the products of nucleic acid amplification procedures (amplicons) have been developed for a variety of solid phases, including nylon membranes, microwell plates, microparticles, and, most recently, microchips (oligonucleotide probe microarrays). This chapter deals with procedures in which the molecules that capture amplicons are immobilized onto the surface of wells of microwell plates. Microwell plate detection systems can be divided into two formats based on the molecule used to capture amplicons: an oligonucleotide probe (sequence-specific capture) and avidin (nonspecific capture). Microwell plate detection systems have been developed in-house and are available commercially. Microwell plate detection procedures are frequently based on capture and detection of biotinylated amplicons. This requires that the primer used in the PCR amplification to generate the amplicon strand complementary to the probe be tagged with biotin. The biotin substituent will allow the amplicon to be either captured or detected by avidin (or streptavidin), depending on the detection format. Polystyrene microwell plates are typically used for amplicon detection because of their high DNA binding capabilities. There is substantial literature demonstrating the excellent analytical performance and clinical utility of microwell plate detection systems for PCR amplicon detection. Microwell plate detection of amplicons is flexible, is compatible with virtually any target, and can easily detect multiple pathogens under common hybridization and wash conditions.

Citation: Fan J, Loeffelholz M. 2011. Microwell Plate Detection Systems for Amplicon Detection and Characterization, p 255-260. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch16

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FIGURE 1

Schematic diagrams of microwell plate detection using sandwich (A) and direct (B and C) hybridization formats.

Citation: Fan J, Loeffelholz M. 2011. Microwell Plate Detection Systems for Amplicon Detection and Characterization, p 255-260. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch16
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Tables

Generic image for table
TABLE 1

Conditions for immobilization of DNA probes in microwell plates

Citation: Fan J, Loeffelholz M. 2011. Microwell Plate Detection Systems for Amplicon Detection and Characterization, p 255-260. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch16
Generic image for table
TABLE 2

Microwell plate hybridization conditions

Citation: Fan J, Loeffelholz M. 2011. Microwell Plate Detection Systems for Amplicon Detection and Characterization, p 255-260. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch16
Generic image for table
TABLE 3

Advantages and disadvantages of microwell plate detection systems versus alternatives

Citation: Fan J, Loeffelholz M. 2011. Microwell Plate Detection Systems for Amplicon Detection and Characterization, p 255-260. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch16

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