Using DNA labels gives biological assays an edge

To detect specific proteins or other molecules in single-cell biological samples, assays often tag the targets with fluorophore-labeled antibodies. But such assays are limited in multiplexing – how many different types of targets they can identify in one test – and can only detect these molecules when they exist in significant amounts. A more modern technique can overcome these limitations, but many details about the process remain unknown.

Alden Moss and Amy Herr found these gaps when they tried to switch their hydrogel experiments to using fluorescent DNA. Previous research had either not studied the method in hydrogel – necessary to Moss and Herr’s work – or not studied the method’s potential for detecting smaller amounts of the target molecules.

Moss and Herr immobilized DNA strands in hydrogel and started the reaction that would create fluorescent nucleotide sequences. They found the reaction only reached full completion after two hours – much longer than predicted. This kind of slowed diffusion had not been seen before, but this reaction had not been thoroughly studied in hydrogels. They also demonstrated increasing the spacing between labeled nucleotides can amplify the fluorescence signal to improve sensitivity, allowing an assay to detect much smaller levels of a given target.

“It’s not just our system that relies on the hydrogel-based DNA readout,” Moss said. “This provides a jumping-off point for assay design principles by identifying this previously unobserved retarded diffusion for this type of process.”

Next, Moss and Herr plan to further characterize the reaction by tracking the polymerase that synthesizes the DNA labels, rather than just the reaction products. They also want to study the reaction in different substrates and find out if more complex systems slow the reaction further.

Source: “In-gel fluorescence detection by DNA polymerase elongation,” by Alden C. Moss and Amy E. Herr, APL Bioengineering (2020). The article can be accessed at https://doi.org/10.1063/5.0021149 .