KAPA hgDNA Quantification and QC Kits contain all the consumables needed for the qPCR-based quantification and quality assessment of human genomic DNA samples prior to NGS library construction. Each kit contains KAPA SYBR FAST qPCR Master Mix, optimized for high-performance SYBR Green I-based qPCR through our Directed Evolution Technology, a set of pre-diluted DNA standards and primer premixes targeting different portions of a highly conserved single-copy human locus. Absolute quantification is achieved with the primer pair defining the shortest fragment and the additional primers providing information about the amount of amplifiable template in the DNA sample. Quality scores (or Q-ratios) generated with the kit may be used to predict the outcome of library construction or tailor workflows for samples of variable quality, particularly FFPE DNA.
First, a single set of DNA standards is used to generate up to three standard curves using three different primer pairs that amplify targets of 41 bp, 129 bp, or 305 bp within a conserved, single-copy human locus. Next, the 41 bp assay is used for absolute quantification of DNA samples. For assessing DNA quality, standard curves are generated and samples assayed with the 129 bp and/or 305 bp primer premix(es). Since poor DNA quality has a greater impact on the amplification of longer targets, the relative quality of a DNA sample can be inferred by normalizing the concentration obtained using the 129 bp or 305 bp assay against the concentration obtained from the 41 bp assay. This normalization generates a Q-ratio (with a value between 0 and 1) that is indicative of DNA quality, or the amount of amplifiable material in a DNA sample.
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