Colborn James M, Byrd Brian D, Koita Ousmane A, Krogstad Donald J
Department of Tropical Medicine, and the Center for Infectious Diseases, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, USA.
Am J Trop Med Hyg. 2008 Dec;79(6):887-92.
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.
虽然SYBR Green用于估计拷贝数，但其荧光会随扩增子长度和腺嘌呤/胸腺嘧啶（AT）含量而变化。因此，使用实时聚合酶链反应（PCR）获得的熔解曲线分析（Ct）值对于较长的扩增子（P<0.001）和具有更高AT含量的扩增子（P<0.001）会更低。相比之下，扩增子长度和AT含量均不影响使用TaqMan探针或LUX标记引物时的Ct值。由于SYBR Green对富含AT的模板和较长的模板产生较低的Ct值，因此它会高估这些模板的拷贝数。因此，如果产生的扩增子富含AT或长度不同，那么在使用实时PCR估计拷贝数时，应考虑使用序列特异性方法，如TaqMan探针或LUX标记引物。
