PCR Technology

0427984 - BTÚ 2015 RIV US eng M - Monography Chapter
Švec, David - Rusňáková, Vendula - Korenková, Vlasta - Kubista, Mikael
Dye-Based High-Throughput qPCR in Microfluidic Platform BioMark.
Washington. In: PCR Technology. Washington: CRC Press Inc, 2013 - (Nolan, T.), s. 323-339. ISBN 9781439848050
Institutional support: RVO:86652036
Keywords : qPCR * microfluidics * fluorescence
Subject RIV: EB - Genetics ; Molecular Biology

When using the dynamic array, each sample is mixed with each assay combinatorially in the integrated fluidic circuits resulting in 48 × 48 = 2304 or 96 × 96 = 9216 qPCR reactions. All the reactions are run in parallel under identical thermal and optical conditions; hence, no interplate calibration is needed and less bias is introduced. When the first BioMark dynamic array was launched in 2007 hydrolysis probes were used exclusively for gene expression analysis, but the advantages of dsDNA binding dye based analysis are in some cases essential: primer design and assay validation process is less complex, in many academic cases the recent primer libraries can be used directly, melt profiles of PCR products can be recorded after each run, and gene expression profiling experiments costs are lower. We developed a temperature protocol for BioMark, which reflects the specific attributes of its optical system together with high background fluorescence of the dsDNA binding dyes in room temperature. We also identified absorbtion problems in the microfluidic environment for some combinations of dye and mastermix and screened several dsDNA binding dyes to find a best solution to achieve robust and reliable high-throughput qPCR analysis
Permanent Link: http://hdl.handle.net/11104/0236442