Plant Cell Reports

, Volume 23, Issue 5, pp 263–271 | Cite as

Use of real-time PCR for determining copy number and zygosity in transgenic plants

  • Ben Bubner
  • Ian T. BaldwinEmail author


This review examines how real-time PCR can be used to determine copy number and zygosity in transgenic plants. Distinguishing between plants that harbor one and two copies of a transgene or are hemizygous and homozygous requires the ability to routinely distinguish twofold differences, a detection difference which approaches the resolution of PCR-based quantification methods. After explaining the basic principles, especially the threshold cycle (Ct value) as the basic measuring unit in real-time PCR, we introduce three quantitation methods currently in use. While the absolute and relative standard curve approaches are qualitative methods that distinguish high-copy from low-copy transformants, the comparative (\(2^{{ - \Delta \Delta {\text{Ct}}}} \)) method with double-dye oligonucleotides (TaqMan probes) is able to detect twofold differences. In order to obtain reliable results, Ct values for an amplicon should be below 25 and the standard deviation below 0.3. Although real-time PCR can deliver exact copy number determinations, the procedure is not fail-safe. Therefore, real-time PCR should to be viewed as complementary to—rather than as a replacement of—other methods such as Southern analysis, but it is particularly useful as a preliminary screening tool for estimating copy numbers of a large number of transformants.


Transgenic plants Real-time PCR Copy number Zygosity 


  1. Bhalla PL, Smith N (1998) Agrobacterium tumefaciens-mediated transformation of cauliflower, Brassica oleracea var. botrytis. Mol Breed 4:531–541CrossRefGoogle Scholar
  2. Bhat SR, Srinivasan S (2002) Molecular and genetic analyses of transgenic plants: considerations and approaches. Plant Sci 163:673–681CrossRefGoogle Scholar
  3. Bieche I, Olivi M, Champeme MH, Vidaud D, Lidereau R, Vidaud M (1998) Novel approach to quantitative polymerase chain reaction using real-time detection: application to the detection of gene amplification in breast cancer. Int J Cancer 78:661–666CrossRefPubMedGoogle Scholar
  4. Bubner B, Gase K, Baldwin IT (2004) Twofold differences are the detection limit for determining transgene copy numbers in plants by real-time PCR. BMC Biotechnol 4:14CrossRefPubMedGoogle Scholar
  5. Callaway AS, Abranches R, Scroggs J, Allen GC, Thompson WF (2002) High-throughput transgene copy number estimation by competitive PCR. Plant Mol Biol Rep 20:265–277Google Scholar
  6. Caplin BE, Rasmussen RP, Bernard PS, Wittwer CT (1999) LightCycler hybridisation probes—the most direct way to monitor PCR amplification and mutation detection. Biochemica 1:5–8Google Scholar
  7. Cullen DW, Lees AK, Toth IK, Duncan JM (2002) Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR. Plant Pathol 51:281–292CrossRefGoogle Scholar
  8. Freeman WM, Walker SJ, Vrana KE (1999) Quantitative RT-PCR: pitfalls and potential. Biotechniques 26:112–125PubMedGoogle Scholar
  9. German MA, Kandel-Kfir M, Swarzberg D, Matsevitz T, Granot D (2003) A rapid method for the analysis of zygosity in transgenic plants. Plant Sci 164:183–187CrossRefGoogle Scholar
  10. Ginzinger DG (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 30:503–512CrossRefPubMedGoogle Scholar
  11. Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real-time quantitative PCR. Genome Res 6:986–994PubMedGoogle Scholar
  12. Hernandez M, Pla M, Esteve T, Prat S, Puigdomenech P, Ferrando A (2003) A specific real-time quantitative PCR detection system for event MON810 in maize YieldGard based on the 3′-transgene integration sequence. Transgenic Res 12:179–189CrossRefPubMedGoogle Scholar
  13. Hernandez M, Esteve T, Prat S, Pla M (2004) Development of real-time PCR systems based on SYBR Green I, Amplifluor and TaqMan technologies for specific quantitative detection of the transgenic maize event GA21. J Cereal Sci 39:99–107CrossRefGoogle Scholar
  14. Holck A, Va M, Didierjean L, Rudi K (2002) 5′-Nuclease PCR for quantitative event-specific detection of the genetically modified Mon810 MaisGard maize. Eur Food Res Technol 214:449–453CrossRefGoogle Scholar
  15. Honda M, Muramoto Y, Kuzuguchi T, Sawano S, Machida M, Koyama H (2002) Determination of gene copy number and genotype of transgenic Arabidopsis thaliana by competitive PCR. J Exp Bot 53:1515–1520CrossRefPubMedGoogle Scholar
  16. Ingham DJ, Beer S, Money S, Hansen G (2001) Quantitative real-time PCR assay for determining transgene copy number in transformed plants. Biotechniques 31:132–140PubMedGoogle Scholar
  17. James VA, Avart C, Worland B, Snape JW, Vain P (2002) The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants. Theor Appl Genet 104:553–561CrossRefPubMedGoogle Scholar
  18. Johnson MR, Wang KS, Smith JB, Heslin MJ, Diasio RB (2000) Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction. Anal Biochem 278:175–184CrossRefPubMedGoogle Scholar
  19. Kim MK, Jeon JH, Fujita M, Davin LB, Lewis NG (2002) The western red cedar (Thuja plicata) 8–8′ DIRIGENT family displays diverse expression patterns and conserved monolignol coupling specificity. Plant Mol Biol 49:199–214CrossRefPubMedGoogle Scholar
  20. Kok JB de, Wiegerinck ETG, Giesendorf BAJ, Swinkels DW (2002) Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes). Hum Mutat 19:554–559CrossRefPubMedGoogle Scholar
  21. Krügel T, Lim M, Gase K, Halitschke R, Baldwin IT (2002) Agrobacterium-mediated transformation of Nicotiana attenuata, a model ecological expression system. Chemoecology 12:177–183Google Scholar
  22. Kutyavin IV, Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES, Singer MJ, Walburger DK, Lokhov SG, Gall AA, Dempcy R, Reed MW, Meyer RB, Hedgpeth J (2000) 3′-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res 28:655–661CrossRefPubMedGoogle Scholar
  23. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  24. Mason G, Provero P, Varia AM, Acotto GP (2003) Estimating the number of integrations in transformed plants by quantitative real-time PCR. BMC Biotechnol 2:20CrossRefGoogle Scholar
  25. McGarvey P, Kaper JM (1991) A simple and rapid method for screening transgenic plants using the PCR. Biotechniques 11:428–432PubMedGoogle Scholar
  26. Meyer P (1998) Stabilities and instabilities in transgene expression. In: Lindsey K (ed) Transgenic plant research. Harwood Academic, Amsterdam, pp 263–275Google Scholar
  27. Negrotto D, Jolley M, Beer S, Wenck AR, Hansen G (2000) The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Rep 19:798–803CrossRefGoogle Scholar
  28. Panchuk II, Volkov RA, Schoffl F (2002) Heat stress- and heat shock transcription factor-dependent expression and activity of ascorbate peroxidase in Arabidopsis. Plant Physiol 129:838–853CrossRefPubMedGoogle Scholar
  29. Ponchel F et al (2003) Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC Biotechnol 3:18CrossRefPubMedGoogle Scholar
  30. ABI PRISM (1997) Sequence Detection System 7700 User Bulletin, Foster City, Calif. No. 2:3–10Google Scholar
  31. ABI PRISM (1998) Sequence Detection System 7700 User Bulletin, Foster City, Calif. No. 5:2–4Google Scholar
  32. Raggi CC et al (1999) Real-time quantitative PCR for the measurement of MYCN amplification in human neuroblastoma with the TaqMan detection system. Clin Chem 45:1918–1924PubMedGoogle Scholar
  33. Robinson JK, Mueller R, Filippone L (2000) New molecular beacon technology. Am Lab 32:30–34Google Scholar
  34. Salmon MA, Vendrame M, Kummert J, Lepoivre P (2002) Detection of apple chlorotic leaf spot virus using a 5′ nuclease assay with a fluorescent 3′ minor groove binder-DNA probe. J Virol Methods 104:99–106CrossRefPubMedGoogle Scholar
  35. Schmidt MA, Parrott WA (2001) Quantitative detection of transgenes in soybean [Glycine max (L.) Merrill] and peanut (Arachis hypogaea L.) by real-time polymerase chain reaction. Plant Cell Rep 20:422–428CrossRefGoogle Scholar
  36. Selvapandiyan A, Reddy VS, Kumar PA, Tewari KK, Bhatnagar RK (1998) Transformation of Nicotiana tabacum with a native cry1Ia5 gene confers complete protection against Heliothis armigera. Mol Breed 4:473–478CrossRefGoogle Scholar
  37. Shou HX, Frame BR, Whitham SA, Wang K (2004) Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation. Mol Breed 13:201–208CrossRefGoogle Scholar
  38. Smith N, Kilpatrick JB, Whitelam GC (2001) Superfluous transgene integration in plants. Crit Rev Plant Sci 20:215–249CrossRefGoogle Scholar
  39. Song P, Cai CQ, Skokut M, Kosegi BD, Petolino JF (2002) Quantitative real-time PCR as a screening tool for estimating transgene copy number in WHISKERS-derived transgenic maize. Plant Cell Rep 20:948–954CrossRefGoogle Scholar
  40. Svensson AS, Johnsson FI, Moller IM, Rasmusson AG (2002) Cold stress decreases the capacity for respiratory NADH oxidation in potato leaves. FEBS Lett 517:79–82CrossRefPubMedGoogle Scholar
  41. Terry CF, Harris N (2001) Event-specific detection of Roundup Ready Soya using two different real time PCR detection chemistries. Eur Food Res Technol 213:425–431CrossRefGoogle Scholar
  42. Tesson L, Heslan JM, Menoret S, Anegon I (2002) Rapid and accurate determination of zygosity in transgenic animals by real-time quantitative PCR. Transgenic Res 11:43–48CrossRefPubMedGoogle Scholar
  43. Weller SA, Elphinstone JG, Smith NC, Boonham N, Stead DE (2000) Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay. Appl Environ Microbiol 66:2853–2858CrossRefPubMedGoogle Scholar
  44. Whitcombe D, Theaker J, Guy SP, Brown T, Little S (1999) Detection of PCR products using self-probing amplicons and fluorescence. Nat Biotechnol 17:804–807CrossRefPubMedGoogle Scholar
  45. Wilhelm J, Pingoud A (2003) Real-time polymerase chain reaction. Chembiochem 4:1120–1128CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.Max-Planck-Institut für Chemische ÖkologieJenaGermany

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