Protocol

DNA and RNA Profiling in Human Blood

Volume 496 of the series METHODS IN MOLECULAR BIOLOGY™ pp 161-174

Nanoliter High-Throughput PCR for DNA and RNA Profiling

  • Colin J. H. BrenanAffiliated withBioTrove Inc.
  • , Douglas RobertsAffiliated withBioTrove Inc.
  • , James HurleyAffiliated withBioTrove Inc.

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Abstract

The increasing emphasis in life science research on utilization of genetic and genomic information underlies the need for high-throughput technologies capable of analyzing the expression of multiple genes or the presence of informative single nucleotide polymorphisms (SNPs) in large-scale, population-based applications. Human disease research, disease diagnosis, personalized therapeutics, environmental monitoring, blood testing, and identification of genetic traits impacting agricultural practices, both in terms of food quality and production efficiency, are a few areas where such systems are in demand. This has stimulated the need for PCR technologies that preserves the intrinsic analytical benefits of PCR yet enables higher throughputs without increasing the time to answer, labor and reagent expenses and workflow complexity. An example of such a system based on a high-density array of nanoliter PCR assays is described here. Functionally equivalent to a microtiter plate, the nanoplate system makes possible up to 3,072 simultaneous end-point or real-time PCR measurements in a device, the size of a standard microscope slide. Methods for SNP genotyping with end-point TaqMan PCR assays and quantitative measurement of gene expression with SYBR Green I real-time PCR are outlined and illustrative data showing system performance is provided.

Key Words

5′-exonuclease assay TaqMan PCR SNP genotyping nanofluidic high-throughput genotyping SYBR Green I real-time PCR nanoliter PCR quantitative PCR