Estimation of copy number using SYBR Green: confounding by AT-rich DNA and by variation in amplicon length.
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.
Colborn
James M
JM
Department of Tropical Medicine, and the Center for Infectious Diseases, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, USA. fue6@cdc.hhs.gov
Byrd
Brian D
BD
Koita
Ousmane A
OA
Krogstad
Donald J
DJ
D000225:Adenine / Q000737:chemistry; D000818:Animals; D000852:Anopheles; D001483:Base Sequence; D052160:Benzothiazoles; D004247:DNA / Q000737:chemistry; D016054:DNA, Protozoan / Q000737:chemistry*; D003959:Diamines; D005260:Female; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D009930:Organic Chemicals / Q000737:chemistry*; D010963:Plasmodium falciparum / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012044:Regression Analysis; D012680:Sensitivity and Specificity; D013941:Thymine / Q000737:chemistry
D052160:Benzothiazoles; D016054:DNA, Protozoan; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA; D000225:Adenine; D013941:Thymine