Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes.
An important consideration in the design of oligonucleotide probes for homogeneous hybridization assays is the efficiency of energy transfer between the fluorophore and quencher used to label the probes. We have determined the efficiency of energy transfer for a large number of combinations of commonly used fluorophores and quenchers. We have also measured the quenching effect of nucleotides on the fluorescence of each fluorophore. Quenching efficiencies were measured for both the resonance energy transfer and the static modes of quenching. We found that, in addition to their photochemical characteristics, the tendency of the fluorophore and the quencher to bind to each other has a strong influence on quenching efficiency. The availability of these measurements should facilitate the design of oligonucleotide probes that contain interactive fluorophores and quenchers, including competitive hybridization probes, adjacent probes, TaqMan probes and molecular beacons.
Marras
Salvatore A E
SA
Public Health Research Institute, 225 Warren Street, Newark, NJ 07103, USA. marras@phri.org
Kramer
Fred Russell
FR
Tyagi
Sanjay
S
D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004735:Energy Transfer; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009711:Nucleotides / Q000378:metabolism; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
D018076:DNA, Complementary; D005456:Fluorescent Dyes; D009711:Nucleotides; D015345:Oligonucleotide Probes