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A comprehensive model for the diffusion and hybridization processes of nucleic acid probes in fluorescence in situ hybridization.
Biotechnology and Bioengineering ( IF 3.5 ) Pub Date : 2020-06-17 , DOI: 10.1002/bit.27462 Joana F Lima 1, 2, 3, 4 , Paulo Maia 1 , Beatriz T Magalhães 1 , Laura Cerqueira 1, 2 , Nuno F Azevedo 1
Biotechnology and Bioengineering ( IF 3.5 ) Pub Date : 2020-06-17 , DOI: 10.1002/bit.27462 Joana F Lima 1, 2, 3, 4 , Paulo Maia 1 , Beatriz T Magalhães 1 , Laura Cerqueira 1, 2 , Nuno F Azevedo 1
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Fluorescence in situ hybridization (FISH) has been extensively used in the past decades for the detection and localization of microorganisms. However, a mechanistic approach of the whole FISH process is still missing, and the main limiting steps for the hybridization to occur remain unclear. In here, FISH is approached as a particular case of a diffusion‐reaction kinetics, where molecular probes (MPs) move from the hybridization solution to the target RNA site within the cells. Based on literature models, the characteristic times taken by different MPs to diffuse across multiple cellular barriers, as well as the reaction time associated with the formation of the duplex molecular probe‐RNA, were estimated. Structural and size differences at the membrane level of bacterial and animal cells were considered. For bacterial cells, the limiting step for diffusion is likely to be the peptidoglycan layer (characteristic time of 7.94 × 102 – 4.39 × 103 s), whereas for animal cells, the limiting step should be the diffusion of the probe through the bulk (1.8–5.0 s) followed by the diffusion through the lipid membrane (1 s). The information provided here may serve as a basis for a more rational development of FISH protocols in the future.
中文翻译：
荧光原位杂交中核酸探针扩散和杂交过程的综合模型。
荧光原位杂交 (FISH) 在过去几十年中被广泛用于微生物的检测和定位。然而，整个 FISH 过程的机制方法仍然缺失，杂交发生的主要限制步骤仍不清楚。在这里，FISH 被视为扩散反应动力学的一个特例，其中分子探针 (MP) 从杂交溶液移动到细胞内的目标 RNA 位点。基于文献模型，估计了不同 MP 扩散穿过多个细胞屏障所需的特征时间，以及与双链分子探针 RNA 形成相关的反应时间。考虑了细菌和动物细胞膜水平的结构和大小差异。对于细菌细胞，2 – 4.39 × 10 3 s)，而对于动物细胞，限制步骤应该是探针通过本体扩散 (1.8–5.0 s)，然后扩散通过脂质膜 (1 s)。此处提供的信息可作为未来更合理开发 FISH 协议的基础。
更新日期：2020-06-17
中文翻译：
荧光原位杂交中核酸探针扩散和杂交过程的综合模型。
荧光原位杂交 (FISH) 在过去几十年中被广泛用于微生物的检测和定位。然而，整个 FISH 过程的机制方法仍然缺失，杂交发生的主要限制步骤仍不清楚。在这里，FISH 被视为扩散反应动力学的一个特例，其中分子探针 (MP) 从杂交溶液移动到细胞内的目标 RNA 位点。基于文献模型，估计了不同 MP 扩散穿过多个细胞屏障所需的特征时间，以及与双链分子探针 RNA 形成相关的反应时间。考虑了细菌和动物细胞膜水平的结构和大小差异。对于细菌细胞，2 – 4.39 × 10 3 s)，而对于动物细胞，限制步骤应该是探针通过本体扩散 (1.8–5.0 s)，然后扩散通过脂质膜 (1 s)。此处提供的信息可作为未来更合理开发 FISH 协议的基础。
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