Sensitivity comparison of real-time PCR probe designs on a model DNA plasmid |
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Authors: | Wang L Blasic J R Holden M J Pires R |
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Institution: | Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA. lili.wang@nist.gov |
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Abstract: | 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. |
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Keywords: | Quantitative real-time PCR Probe design TaqMan Molecular beacon Shared-stem molecular beacon DNA plasmid Detection sensitivity Signal-to-background ratio 5′ Exonuclease activity Fluorescence quantum yield Melting temperature |
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