Influence of Nucleotide-biased Fluorescence Emissions of SYBR Green Ⅱ on the Result Consistence of Rolling Circle Amplification

The fluorescence dye SYBR Green Ⅱ(SG Ⅱ)has been frequently used in rolling circle amplification(RCA)based analyses of nucleic acids.However,a good amount of inconsistencies have been reported in regards the quality and reproducibility of RCA reactions.To properly examine this experimental issue,here we utilized a series of synthetic oligonucleotides and circular templates to investigate the impact of SG Ⅱ in RCA reactions.The results indicate that SG Ⅱ enables a strong fluorescence signal only when complexing with guanosine(G)residue.In RCA reactions,long single-stranded RCA products,enriched with G residues,result in higher fluorescence emission when compared with the addition of other nucleotide residues.These results suggest that the nucleotide composition of the reaction can affect the amplification results and,eventually,can lead to inconsistent fluorescence of the RCA products.This work indicates that particular attention should be given when circular templates are designed for the quantitative analysis of nucleic acids,to further allow the signal reproducibility of RCA-based experiments.