New FRET primers for quantitative real-time PCR
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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.
KeywordsReal-time PCR FRET Primers Labeled primers Quantitative real-time PCR FAM Texas red
Financial support from Chalmers University of Technology is gratefully acknowledged.
- 4.Wittwer CT, Ririe KM, Andrew RV, David DA, Gundry RA, Balis UJ (1997) Biotechniques 22:176–181Google Scholar
- 5.Morrison TM, Weis JJ, Wittwer CT (1998) Biotechniques 24:954–962Google Scholar
- 6.Ahmad A (2007) J Biosciences, (in press)Google Scholar
- 10.Livak K, Flood S, Marmaro J, Giusti W, Deetz K (1995) PCR Methods Appl 4:357–362Google Scholar
- 12.Caplin BE, Rasmussen RP, Bernar PS, Wittwer CT (1999) Biochemica 1:5–8Google Scholar
- 14.Irina AA, Sylvia S, David W, Yevgeniy SB (2002) PharmaGenomics 2:48–54Google Scholar
- 15.Irina AA, Michael WR, Lusby E, Irina GS, Yevgeniy SB (2002) Biotechniques 32:940–949Google Scholar
- 19.Islam A, Hazra P (2003) Int patent WO 03/102239 A2Google Scholar