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

Biotechniques. 2020 Aug;69(2):80-83. doi: 10.2144/btn-2020-0016. Epub 2020 Jun 5.

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.

Keywords: hook effect; optimization; quantitation; quantitation artifact; quantitative PCR.

Publication types

  • Letter
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Artifacts
  • DNA* / analysis
  • DNA* / chemistry
  • DNA* / metabolism
  • Fluorescent Dyes* / analysis
  • Fluorescent Dyes* / chemistry
  • Intercalating Agents / analysis
  • Intercalating Agents / chemistry
  • Real-Time Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Temperature

Substances

  • Fluorescent Dyes
  • Intercalating Agents
  • DNA