Mechanism And Application For Quenching Effect Of Bases On Fluorophore

Part I Mechanism and regularity for quenching effect of bases on fluorophore Objective: To elucidate the mechanism of the base-quenched probe method.Methods: We investigated the possible mechanism of fluorescence quenching by DNA bases in aqueous solution using spectroscopic techniques.Next,we analyzed electron transfer or transmission between DNA bases and fluorophores.Results: It showed that the possible mechanism of the base-quenched probe method might be photo-induced electron transfer.Moreover,the data suggested that in single-stranded DNA,the electrons of the fluorophore are transferred to the orbit of pyrimidine bases(Thymine(T)and cytosine(C)),or that the electron orbits of the fluorophore are occupied by electrons from purine bases(Guanine(G)and adenine(A)),which lead to fluorescence quenching.In addition,the electrons of a fluorophore excited by light can be transmitted along double-stranded DNA,which gives rise to stronger fluorescence quenching.Furthermore,we demonstrated that the quenching efficiency of bases is in the order of G > C ≥ A ≥ T and the capability of electron transmission of base-pairs in double-stranded DNA is in the order of CG ≥ GC > TA ≥ AT(letters representing bases on the complementary strand of the probe are bold and underlined),and the most common commercial fluorophores including FAM,HEX,TET,JOE,and TAMRA could be influenced by bases and are in line with this mechanism and regularity.Conclusion: The mechanism by which bases quench fluorophore is electron transfer and transmission in DNA strand.Part II Detection of simultaneous multi-mutations using base-quenched probeObjective: To optimize and improve the base-quenched probe method and to establish a method for detecting multi-mutations in a single tube.Methods: We applied the most common commercial fluorescent dyes including FAM,HEX,CY5,CY3,TET,JOE,Texas Red and ROX for labeling probes.Each fluorescent dye’s interference pattern was revealed by PCR together with melting curve analyses for detecting multi-mutations simultaneously according to the different fluorescence channels.Accuracy of the method was confirmed by direct sequencing.Results: With the temperature increases,the fluorescence of HEX,ROX,Texas Red and TET are hardly influenced,while the fluorescence of FAM,JOE,CY3 and Cy5 are decreased.FAM,HEX,CY5,CY3,TET,JOE,Texas Red,or ROX could be influenced by bases and could be applied to detect single nucleotide polymorphism.According to the different characteristics of fluorophores,this method was applied to detect APOM rs707921,APOM rs707922 and MCP-1 rs1024611 simultaneously,which was demonstrated successful.Conclusion: The method for detecting multi-mutations in a single tube is simple,rapid and accurate and is suitable for a large-scale genotype screening.