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Target sequence heterogeneity causes the 'hook effect' in fluorescent dye-based quantitative PCR

Abstract

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.

Type Letter
ISBN 1940-9818 (Electronic) 0736-6205 (Linking)
Authors Warton, K.; Xu, Y.; Ford, C. E.
Responsible Garvan Author Dr Kristina Warton
Publisher Name BIOTECHNIQUES
Published Date 2020-08-01
Published Volume 69
Published Issue 2
Published Pages 80-83
Status Published in-print
DOI 10.2144/btn-2020-0016
URL link to publisher's version https://www.ncbi.nlm.nih.gov/pubmed/32500726