Sensitivity comparison of real-time PCR probe designs on a model DNA plasmid.
We investigated three probe design strategies used in quantitative polymerase chain reaction (PCR) for sensitivity in detection of the PCR amplicon. A plasmid with a 120-bp insert served as the DNA template. The probes were TaqMan, conventional molecular beacon (MB), and shared-stem molecular beacon (ATssMB and GCssMB). A shared-stem beacon probe combines the properties of a TaqMan probe and a conventional molecular beacon. It was found that the overall sensitivities for the four PCR probes are in the order of MB>ATssMB>GCssMB>TaqMan. The fluorescence quantum yield measurements indicate that incomplete or partial enzymatic cleavage catalyzed by Taq polymerase is the likely cause of the low sensitivities of two shared-stem beacons when compared with the conventional beacon probe. A high-fluorescence background associated with the current TaqMan probe sequence contributes to the relatively low detection sensitivity and signal-to-background ratio. The study points out that the nucleotide environment surrounding the reporting fluorophore can strongly affect the probe performance in real-time PCR.
Wang
L
L
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA. lili.wang@nist.gov
Blasic
J R
JR
Holden
M J
MJ
Pires
R
R
D001483:Base Sequence; D015342:DNA Probes; D008954:Models, Biological; D008969:Molecular Sequence Data; D043264:Phosphodiesterase I / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D019914:Taq Polymerase / Q000378:metabolism
D015342:DNA Probes; D019914:Taq Polymerase; D043264:Phosphodiesterase I