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Mechanism-based qPCR quantification methods have also been suggested, and have the advantage that they do not require a standard curve for quantification. Methods such as MAK2 [16] have been shown to have equal or better quantitative performance to standard curve methods. These mechanism-based methods use knowledge about the polymerase ...
The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines are a set of protocols for conducting and reporting quantitative real-time PCR experiments and data, as devised by Bustin et al. in 2009. [1]
Besides reporting guidelines, the MIQE stresses the need to standardize the nomenclature associated with quantitative PCR to avoid confusion; for example, the abbreviation qPCR should be used for quantitative real-time PCR, while RT-qPCR should be used for reverse transcription-qPCR, and genes used for normalization should be referred to as ...
The fifth and final step is the analyzation step of the ChIP protocol by the process of qPCR, ChIP-on-chip (hybrid array) or ChIP sequencing. Oligonucleotide adaptors are then added to the small stretches of DNA that were bound to the protein of interest to enable massively parallel sequencing. Through the analysis, the sequences can then be ...
qPCR is unable to distinguish differences in gene expression or copy number variations that are smaller than twofold. On the other hand, dPCR has a higher precision and has been shown to detect differences of less than 30% in gene expression, distinguish between copy number variations that differ by only 1 copy, and identify alleles that occur ...
Chimeric polymerases overcome many limitations of native enzymes and are used in direct PCR amplification from cell cultures and even food samples, thus by-passing laborious DNA isolation steps. A robust strand-displacement activity of the hybrid TopoTaq polymerase helps solve PCR problems that can be caused by hairpins and G-loaded double helices.