Rolling circle amplification with fluorescently labeled dUTP-balancing the yield and degree of labeling

Anal Bioanal Chem. 2021 Apr 8. doi: 10.1007/s00216-021-03322-7. Online ahead of print.ABSTRACTDetection methods based on rolling circle amplification (RCA) have been applied to a large number of targets in molecular biology. The key feature of RCA-based methods as well as other nucleic acid amplification methods is their exceptional sensitivity, which allows the detection of molecules at low concentrations, achieved by signal amplification due to nucleic acid magnification and subsequent detection. Variations on the method, such as immuno-RCA, extend the range of potential targets that can be detected. Employing fluorescently labeled nucleotides for direct incorporation into an amplification product is an attractive method for RCA product detection. However, the effectiveness of this approach remains doubtful. In our study, we utilized different modified dUTPs, including sulfo-cyanine3-dUTP, sulfo-cyanine5-dUTP, sulfo-cyanine5.5-dUTP, BDP-FL-dUTP, and amino-11-dUTP, to investigate whether the properties of the fluorophore used for modification affected the reaction yield and effectiveness of incorporation of nucleotide analogs by phi29 DNA polymerase. Among the modified dUTPs, sulfo-cyanine3-dUTP demonstrated the highest incorporation effectiveness, equal to 4-9 labels per 1000 nucleotides. The mean length of the RCA product was estimated to be approximately 175,000 nucleotides. The total increase in fluorescence from a single target/product complex was 850 times. The results...
Source: Analytical and Bioanalytical Chemistry - Category: Chemistry Authors: Source Type: research