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Estimation of Copy Number using SYBR Green: Confounding by AT-rich DNA and by Variation in Amplicon Length

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  • 1 Departments of Tropical Medicine and Medicine, and the Center for Infectious Diseases, Tulane University Health Sciences Center, New Orleans, Louisiana; Department of Biology, Faculty of Science and Technology, University of Bamako, Bamako, Mali
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Although SYBR Green is used to estimate copy number, its fluorescence varies with amplicon length and adenine/thymine (AT) content. As a result, threshold cycle (Ct) values obtained using real-time polymerase chain reaction (PCR) are lower for longer amplicons (P < 0.001) and amplicons with greater AT content (P < 0.001). In contrast, neither amplicon length nor AT content affects the Ct with TaqMan probes or LUX-labeled primers. Because SYBR Green yields lower Cts with AT-rich templates and longer templates, it overestimates copy number for those templates. Therefore, sequence-specific methods such as TaqMan probes or LUX-labeled primers should be considered when using real-time PCR to estimate copy number if the amplicons generated are AT-rich or vary in length.

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