Homogenous Detection of Nucleic Acids Using Self-Quenched Polymerase Chain Reaction Primers Labeled

Multiplex quantitative polymerase chain reaction (PCR) based on novel design of fluorescent primers is described. Self-quenched 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 nucleotides 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 upon formation of the PCR product. The hairpin oligonucleotides (ΔG from -1.6 to -5.8 kcal/mol) are as efficient as linear primers and provide additional specificity to the PCR by preventing primer-dimers and mispriming. Self-quenched primers could be designed manually or by specialized software and could be used for real-time gene quantitation. Targets of 10-107 copies could be detected with precision in PCR using fluorescein-labeled primers for variable genes and JOE-labeled primers for the reference genes. This method could also be used to detect single nucleotide polymorphism by allele-specific PCR. In conclusion, self-quenched primers are an efficient and cost-effective alternative to fluorescence resonance energy transfer-labeled oligonucleotides.