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Am. J. Trop. Med. Hyg., 79(6), 2008, pp. 887-892
Copyright © 2008 by The American Society of Tropical Medicine and Hygiene

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

James M. Colborn, Brian D. Byrd, Ousmane A. Koita, AND Donald J. Krogstad*
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

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.

Received February 27, 2008. Accepted for publication August 26, 2008.

Acknowledgments: We thank Fawaz Mzayek for assistance with the statistical analysis; Fran Krogstad for cultivation of P. falciparum; the American Type Culture Collection for providing the Anopheles gambiae G3 strain contributed by Mark Q. Benedict; the Sequencing Core of the Tulane Center for Gene Therapy for sequencing cloned block 2 regions of msp1 from K1, MAD20 and hybrid MAD20/RO33 P. falciparum parasites; Young Hong, Kenneth Swan, Maria Morales, Tunika Okatcha, and Haiyan Deng for thoughtful reviews of the manuscript; and Mark Lawson (Bio-Rad) for permission to cite data from their recent survey about the use of SYBR Green for real-time PCR to estimate copy number.

Financial support: These studies were supported in part by a cooperative agreement from the Emerging Infectious Diseases Program of the Centers for Disease Control and Prevention (CDC) (CCU/UR3 418652). Graduate stipends for James M. Colborn and Brian D. Byrd were supported by the Tulane/CDC Vector-borne Infectious Disease Training Fellowship (CDC Cooperative Agreement T01/CCT622308) and by the Emerging Infectious Diseases Program of the CDC (CCU/ UR3 418652 and U01 CI 000211).

* Address correspondence to Donald J. Krogstad, Departments of Tropical Medicine and Medicine, and the Center for Infectious Diseases, Tulane University Health Sciences Center, 1430 Tulane Avenue, SL 17, J. Bennett Johnston Building, Room 510, New Orleans, LA 70112. E-mail: krogstad{at}tulane.edu

Note: Supplemental Table 1 (Sequences of Synthetic DNA Templates), Supplemental Table 2 (Primers and probes used for real-time PCR), Supplemental Table 3 (Data used for the multivariate regression analysis) and Supplemental Figure 1 (Expected Relationship between Changes in Fluorescence (based on AT and GC base pair composition) and Changes in the Threshold Concentration (Ct)) appear online at www.ajtmh.org.

Authors’ addresses: James M. Colborn, Dengue Branch, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, San Juan, PR 00920, E-mail: fue6{at}cdc.hhs.gov. Brian D. Byrd, College of Health and Human Sciences, Western Carolina University, Cullowhee, NC 28723, E-mail: bdbyrd{at}wcu.edu. Ousmane A. Koita, Faculty of Science and Technology, University of Bamako, BP E 3206, Bamako, Mali, E-mails: oakoita{at}yahoo.com and oakoita{at}ml.refer.org. Donald J. Krogstad, Departments of Tropical Medicine and Medicine, and the Center for Infectious Diseases, Tulane University Health Sciences Center, 1430 Tulane Avenue, SL 17, J. Bennett Johnston Building, New Orleans, LA 70112, E-mail: krogstad{at}tulane.edu.






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Copyright © 2008 by the American Society of Tropical Medicine and Hygiene.