Estimation of copy number using SYBR green: Confounding by AT-rich DNA and by variation in amplicon length

Abstract

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 (C1) 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 C1 with TaqMan probes or LUX-labeled primers. Because SYBR Green yields lower C1S 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. Copyright © 2008 by The American Society of Tropical Medicine and Hygiene.