Homogeneous detection of nucleic acids using self-quenched polymerase chain reaction primers labeled with a single fluorophore (LUX primers).
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 (DeltaG 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.
Nazarenko
Irina
I
Research and Development, Digene Corporation, Gaithersburg, MD, USA.
D004247:DNA / Q000032:analysis; D017931:DNA Primers; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes; D015336:Molecular Probe Techniques; D015335:Molecular Probes; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009696:Nucleic Acids / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000032:analysis
D017931:DNA Primers; D005456:Fluorescent Dyes; D015335:Molecular Probes; D009696:Nucleic Acids; D012313:RNA; D004247:DNA