Target sequence heterogeneity causes the 'hook effect' in fluorescent dye-based quantitative PCR
The 'hook effect' describes a phenomenon in quantitative PCR (qPCR) amplification curves where fluorescence values decrease following an initial amplification phase. We propose that in intercalating dye-based qPCR, the 'hook effect' is due to the amplification of heterogeneous but related DNA targets. The decrease in fluorescence at later cycles occurs because the related products self-anneal to form a DNA heteroduplex with a melt temperature below the temperature at which the fluorescence measurement is made. We show this experimentally using qPCR of Alu family repetitive DNA elements.
|ISBN||1940-9818 (Electronic) 0736-6205 (Linking)|
|Authors||Warton, K.; Xu, Y.; Ford, C. E.|
|Responsible Garvan Author|
|URL link to publisher's version||https://www.ncbi.nlm.nih.gov/pubmed/32500726|