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Nucleic Acids Research, 2002, Vol. 30, No. 9 e37
© 2002 Oxford University Press

Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore

Irina Nazarenko*, Brian Lowe, Marlene Darfler, Pranvera Ikonomi, David Schuster and Ayoub Rashtchian

Invitrogen Corporation, 1620 Faraday Avenue, Carlsbad, CA 92008, USA

Multiplex quantitative PCR based on novel design of fluorescent primers is described. Fluorogenic primers are labeled with a single fluorophore on a base close to the 3' end with no quencher required. A tail of 5–7 nt is added to the 5' end of the primer to form a blunt-end hairpin when the primer is not incorporated into a PCR product. This design provides a low initial fluorescence of the primers that increases up to 8-fold upon formation of the PCR product. The hairpin oligonucleotides ({Delta}G from –1.6 to –5.8 kcal/mol) may be as efficient as linear primers and provide additional specificity to the PCR by preventing primer-dimers and mispriming. Multiple fluorogenic primers were designed by specialized software and used for real-time quantitation of c-myc and IL-4 cDNAs in the presence of reference genes such as ß-actin, GAPDH and 18S rRNA. Targets of 10–107 copies were detected with precision in PCR using FAM-labeled primers for variable genes and JOE-labeled primers for the reference genes. This method was also used to detect single nucleotide polymorphism of the human retinal degeneration gene by allele-specific PCR with end-point detection using a fluorescent plate reader or a UV-transilluminator. We conclude that fluorogenic mono-labeled primers are an efficient and cost-effective alternative to FRET-labeled oligonucleotides.

* To whom correspondence should be addressed at present address: 708 Quince Orchard Road, Gaithersburg, MD 20878, USA. Tel: +1 301 987 1711; Fax: +1 301 987 1717; Email: inazarenko{at}metrigenix.com


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