Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore.
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 (DeltaG 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 beta-actin, GAPDH and 18S rRNA. Targets of 10-10(7) 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.
Nazarenko
Irina
I
Invitrogen Corporation, 1620 Faraday Avenue, Carlsbad, CA 92008, USA. inazarenko@metrigenix.com
Lowe
Brian
B
Darfler
Marlene
M
Ikonomi
Pranvera
P
Schuster
David
D
Rashtchian
Ayoub
A
D000483:Alleles; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012333:RNA, Messenger / Q000032:analysis*; D012680:Sensitivity and Specificity; D013997:Time Factors
D017931:DNA Primers; D005456:Fluorescent Dyes; D012333:RNA, Messenger