In recent times, with the development of technologies, there has been a significant increase in single-molecule studies. The single-molecule study has allowed us to know about the biological phenomenon, for example, DNA dynamics, protein-nucleic acid interaction, etc. which led us to find out the distinct molecular behavior which would have been very difficult with the bulk study. It is one of the most important requirements for a single-molecule study is to have a condition where individual molecules can be immobilized without affecting their biological property. Here, we studied the conditions required for the two-end immobilization of the lambda DNA on the PEG functionalized surface. In this approach, the DNA is immobilized at the two ends by making use of the biotin neutravidin interaction. The DNA is labeled at both ends with biotin. PEG surface is terminated with biotin and mixed with methoxy terminated PEG. Afterward, neutravidin is added to the biotin on the surface. This allows for biotinylated DNA to be stretched by fluid flow and binding to the neutravidin on the surface. We optimize the neutravidin distribution on the surface by controlling the biotin PEG to methoxy PEG ratio for DNA immobilization. We also studied the significance of flow rate and ionic strength of the buffer on DNA elongation in the microfluidic channel. Total internal reflection fluorescence (TIRF) microscopy is used to visualize the multiple immobilized DNA on the surface. This will improve DNA immobilization and reduce the nonspecific binding of DNA on the surface.

Optimize the conditions for the two end DNA immobilization on the glass substrate

ACS Spring 2022

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