A comprehensive model for the diffusion and hybridization processes of nucleic acid probes in fluorescence in situ hybridization

• Type: Another Publication in an International Scientific Journal
• Date: 2020

Authors

Lima, JF (Author) Other; Maia, P (Author) Other; Magalhaes, BT (Author) Other; Cerqueira, L (Author) FEUP; Azevedo, NF (Author) FEUP

Journal

Title: Biotechnology and Bioengineering

Vol. 117

Pages: 3212-3223

ISSN: 0006-3592

Publisher: Wiley-Blackwell

Indexing

ISI Web of Knowledge - 2 Citations

Scopus - 2 Citations

Other information

Authenticus ID: P-00S-DFZ

DOI: 10.1002/bit.27462

Abstract (EN): 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 x 10(2) - 4.39 x 10(3) 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.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 12