Use of DNA and peptide nucleic acid molecular beacons for detection and quantification of rRNA in solution and in whole cells
Use of DNA and peptide nucleic acid molecular beacons for detection and quantification of rRNA in solution and in whole cells
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DOI：
10.1128/aem.69.9.5673-5678.2003
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发表时间：
2003-09-01
影响因子：
4.4
通讯作者：
Raskin, L
中科院分区：
文献类型：
--
作者：
Xi, CW;Balberg, M;Raskin, L
DNA和肽核酸（PNA）分子信标被成功地用于检测溶液中的rRNA。此外，发现PNA分子信标杂交可用于定量rRNA：在50 nM PNA MB存在下，杂交信号随本实验中使用的16S rRNA浓度（0.39至25 nM）以线性方式增加。DNA和PNA分子信标被成功地用于在荧光原位杂交（FISH）实验中检测全细胞而无需洗涤步骤。PNA分子信标的FISH结果上级优于DNA分子信标：杂交动力学快得多，信噪比高得多，特异性好得多。最后，证明了在FISH中使用PNA分子信标和细胞术的结合使得快速收集定量FISH数据成为可能。因此，PNA分子信标可能为传统FISH方法的局限性提供解决方案，例如可变的靶位点可达性，对具有低rRNA含量的靶细胞的灵敏度差，背景荧光，以及FISH在微流体装置中的应用。
DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution. In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA MB. DNA and PNA molecular beacons were successfully used to detect whole cells in fluorescence in situ hybridization (FISH) experiments without a wash step. The FISH results with the PNA molecular beacons were superior to those with the DNA molecular beacons: the hybridization kinetics were much faster, the signal-to-noise ratio was much higher, and the specificity was much better for the PNA molecular beacons. Finally, it was demonstrated that the combination of the use of PNA molecular beacons in FISH and How cytometry makes it possible to rapidly collect quantitative FISH data. Thus, PNA molecular beacons might provide a solution for limitations of traditional FISH methods, such as variable target site accessibility, poor sensitivity for target cells with low rRNA content, background fluorescence, and applications of FISH in microfluidic devices.