Synthesis and evaluation of a new non-fluorescent quencher in fluorogenic oligonucleotide probes for real-time PCR.
A non-fluorescent quencher, based on the diaminoanthraquinone Disperse Blue 3, has been incorporated into oligonucleotides at the 5'-end, the 3'-end and internally as a thymidine derivative. Fluorimetry and fluorogenic real-time PCR experiments demonstrate that the quencher is effective with a wide range of fluorescent dyes. The anthraquinone moiety increases the melting temperature of DNA duplexes, thus allowing shorter, more discriminatory probes to be used. The quencher has been used in Scorpion primers and TaqMan probes for human DNA sequence recognition and mutation detection.
School of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK.
Jonathan P
JP
May
Lynda J
LJ
Brown
Ian
I
van Delft
Nicola
N
Thelwell
Kate
K
Harley
Tom
T
Brown
D001391:Azo Compounds / Q000737:chemistry; D001483:Base Sequence; D002863:Chromogenic Compounds / Q000138:chemical synthesis* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000138:chemical synthesis* / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000138:chemical synthesis* / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D013057:Spectrum Analysis
D001391:Azo Compounds; D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D009943:Organophosphorus Compounds; C434331:phosphoramidite; C008492:methyl red