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Analytical and Bioanalytical Chemistry

, Volume 387, Issue 8, pp 2737–2743 | Cite as

New FRET primers for quantitative real-time PCR

  • Ashraf I. Ahmad
  • Jahan B. GhasemiEmail author
Original Paper

Abstract

FRET primer real-time PCR chemistry depends on internally labeled primers with FRET dyes linked to their 3′ end. The best distance between the FRET dyes for obtaining the largest signal and the lowest background is six nucleotides. In this study the forward primer was labeled with FAM and the reverse primer with Texas red; the labeled primers meet in cycle two of PCR. At the end of the elongation step FAM is excited to emit fluorescence which will excite Texas red to emit new fluorescence that correlates directly with the quantity of PCR product accumulated. FRET primer techniques amplify short amplicons with unique thermal cycling steps, 0 s at 85 °C for denaturation, 7 s for annealing, and 2 s for elongation. The FRET primer technique was very efficient (92.6, 97.2, and 100%), correlation coefficients were high (1.0, 0.999, and 0.999), and total run time was very short (20, 45, and 40 min per 40 cycles with LightCycler, iCycler, and RotorGene 3000, respectively). When FRET-labeled primers were compared with similar but unlabeled primers it was observed that the FRET primer technique had a lower Ct value and was more efficient than use of unlabeled primers detected by use of SYBR Green I.

Figure

Schematic diagram of FRET prime real-time PCR

Keywords

Real-time PCR FRET Primers Labeled primers Quantitative real-time PCR FAM Texas red 

Notes

Acknowledgment

Financial support from Chalmers University of Technology is gratefully acknowledged.

Supplementary material

216_2007_1123_MOESM1_ESM.doc (458 kb)
Supplementry Figures (DOC 457 KB)

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Department of Chemical and Biological Engineering—Molecular BiotechnologyChalmers University of TechnologyGöteborgSweden
  2. 2.Department of ChemistryRazi UniversityKermanshahIran

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