Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore
AUTOR(ES)
Nazarenko, Irina
FONTE
Oxford University Press
RESUMO
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 (ΔG 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 β-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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=113860Documentos Relacionados
- Multiplex genotyping of PCR products with MassTag-labeled primers.
- Fingerprinting genomes using PCR with arbitrary primers.
- The production of PCR products with 5' single-stranded tails using primers that incorporate novel phosphoramidite intermediates.
- High-Throughput SNP Genotyping by Allele-Specific PCR with Universal Energy-Transfer-Labeled Primers
- Self-Reporting PNA/DNA Primers for PCR Analysis