Target sequence heterogeneity causes the ‘hook effect’ in fluorescent dye-based quantitative PCR
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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.
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1991 ◽
Vol 42
(2)
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pp. 243-249
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1980 ◽
pp. 217-234
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2001 ◽
Vol 197
(2)
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pp. 139-143
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