Volume 79, Issue 6
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645


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 (C) values obtained using real-time polymerase chain reaction (PCR) are lower for longer amplicons ( < 0.001) and amplicons with greater AT content ( < 0.001). In contrast, neither amplicon length nor AT content affects the C with TaqMan probes or LUX-labeled primers. Because SYBR Green yields lower Cs 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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  1. Schneeberger C, Speiser P, Kury F, Zeillinger R, 1995. Quantitative detection of reverse transcriptase-PCR products by means of a novel and sensitive DNA stain. PCR Methods Appl 4 : 234–238.
  2. Bustin SA, 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25 : 169–193.
  3. Zipper H, Brunner H, Bernhagen J, Vitzthum F, 2004. Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic Acids Res 32 : e103.
  4. Zipper H, Buta C, Lammle K, Brunner H, Bernhagen J, Vitzthum F, 2003. Mechanisms underlying the impact of humic acids on DNA quantification by SYBR Green I and consequences for the analysis of soils and aquatic sediments. Nucleic Acids Res 31 : e39.
  5. Vitzthum F, Geiger G, Bisswanger H, Brunner H, Bernhagen J, 1999. A quantitative fluorescence-based microplate assay for the determination of double-stranded DNA using SYBR Green I and a standard ultraviolet transilluminator gel imaging system. Anal Biochem 276 : 59–64.
  6. Trager W, Jensen JB, 1976. Human malaria parasites in continuous culture. Science 193 : 673–675.
  7. Campbell CC, Spencer HC, Chin W, Collins WE, 1980. Adaptation of cultured Plasmodium falciparum to the intact squirrel monkey (Saimiri sciureus). Trans R Soc Trop Med Hyg 74 : 548–549.
  8. Teklehaimanot A, Nguyen-Dinh P, Collins WE, Barber AM, Campbell CC, 1985. Evaluation of sporontocidal compounds using Plasmodium falciparum gametocytes produced in vitro. Am J Trop Med Hyg 34 : 429–434.
  9. Mabuchi T, Nishikawa S, 1990. Selective staining with two fluorochromes of DNA fragments on gels depending on their AT content. Nucleic Acids Res 18 : 7461–7462.
  10. Molecular Probes Product Information, (Revision July 1, 2005). OliGreen ssDNA Quantitation Reagent and Kits. Available at: http://probes.invitrogen.com/media/pis/mp07582.pdf.
  11. Rosen S, Skaletsky H, 2000. Primer3 on the WWW for General Users and for Biologist Programmers. Krawetz SA, Misener S, eds. Bioinformatics Methods and Protocols: Methods in Molecular Biology. Totowa, NJ: Humana Press, 365–386.
  12. Blazquez S, Zimmer C, Guigon G, Olivo-Marin JC, Guillen N, Labruyere E, 2006. Human tumor necrosis factor is a chemo-attractant for the parasite Entamoeba histolytica. Infect Immun 74 : 1407–1411.
  13. Besansky NJ, Bedell JA, Benedict MQ, Mukabayire O, Hilfiker D, Collins FH, 1995. Cloning and characterization of the white gene from Anopheles gambiae. Insect Mol Biol 4 : 217–231.
  14. Luna C, Wang X, Huang Y, Zhang J, Zheng L, 2002. Characterization of four Toll related genes during development and immune responses in Anopheles gambiae. Insect Biochem Mol Biol 32 : 1171–1179.
  15. Weller P, Jeffreys AJ, Wilson V, Blanchetot A, 1984. Organization of the human myoglobin gene. EMBO J 3 : 439–446.
  16. Koita O, 2000. Molecular Studies of Clonality, Transmission and Severe Disease in Malaria. PhD dissertation. New Orleans, LA: Tulane University. Available from ProQuest Dissertations and Theses, Ann Arbor, MI. Accessed August 19, 2008. Publication no. AAT 9997229.
  17. Ferreira MU, Liu Q, Kaneko O, Kimura M, Tanabe K, Kimura EA, Katzin AM, Isomura S, Kawamoto F, 1998. Allelic diversity at the merozoite surface protein-1 locus of Plasmodium falciparum in clinical isolates from the southwestern Brazilian Amazon. Am J Trop Med Hyg 59 : 474–480.
  18. Vaerman JL, Saussoy P, Ingargiola I, 2004. Evaluation of real-time PCR data. J Biol Regul Homeost Agents 18 : 212–214.
  19. Gudnason H, Dufva M, Bang DD, Wolff A, 2007. Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic Acids Res 35 : e127.
  20. Giglio S, Monis PT, Saint CP, 2003. Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR. Nucleic Acids Res 31 : e136.
  21. Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S, Paulsen IT, James K, Eisen JA, Rutherford K, Salzberg SL, Craig A, Kyes S, Chan M-S, Nene V, Shallom S, Suh B, Peterson J, Angiuoli S, Pertea M, Allen J, Selengut J, Haft D, Mather MW, Vaidya AB, Martin DM, Fairlamb AH, Fraunholz MJ, Roos DS, Ralph SA, McFadden GI, Cummings LM, Subramanian GM, Mungall C, Venter JC, Carucci DJ, Hoffman SL, Newbold C, Davis RW, Fraser CM, Barrell B, 2002. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419 : 498–511.
  22. Glockner G, Eichinger L, Szafranski K, Pachebat JA, Bankier AT, Dear PH, Lehmann R, Baumgart C, Parra G, Abril JF, Guigo R, Kumpf K, Tunggal B, Cox E, Quail MA, Platzer M, Rosenthal A, Noegel AA, and the Dictyostelium Genome Sequencing Consortium, 2002. Sequence and analysis of chromosome 2 of Dictyostelium discoideum. Nature 418 : 79–85.
  23. Goebel SJ, Johnson GP, Perkus ME, Davis SW, Winslow JP, Paoletti E, 1990. The complete DNA sequence of vaccinia virus. Virology 179: 247–266, 517–563.
  24. Pridmore RD, Berger B, Desiere F, Vilanova D, Barretto C, Pittet AC, Zwahlen MC, Rouvet M, Altermann E, Barrangou R, Mollet B, Mercenier A, Klaenhammer T, Arigoni F, Schell MA, 2004. The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533. Proc Natl Acad Sci USA 101 : 2512–2517.
  25. Westberg J, Persson A, Holmberg A, Goesmann A, Lundeberg J, Johansson KE, Pettersson B, Uhlen M, 2004. The genome sequence of Mycoplasma mycoides subsp. mycoides SC type strain PG1T, the causative agent of contagious bovine pleuropneumonia (CBPP). Genome Res 14 : 221–227.
  26. Goulter AB, Harmer DW, Clark KL, 2006. Evaluation of low density array technology for quantitative parallel measurement of multiple genes in human tissue. BMC Genomics 7 : 34.
  27. Ferreira ID, Rosario VE, Cravo PV, 2006. Real-time quantitative PCR with SYBR Green I detection for estimating copy numbers of nine drug resistance candidate genes in Plasmodium falciparum. Malar J 5 : 1.

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Supplementary Data

Supplemental tables and figure

  • Received : 27 Feb 2008
  • Accepted : 26 Aug 2008

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