Application Of Single Strand Specific Nuclease S1 And 2’F Nucleotide To Improve The Specificity Of Nucleic Acid Detection

Objective: To investigate the role of S1 and 2’F nucleotide derivatives of single chain specific nuclease in enhancing the specificity of nucleic acid thermostatic detection.Some mi RNAs can be used as biomarkers for tumor diagnosis and prognosis prediction,such as let7 mi RNA,mi RNA200 family,etc.,but their family homology is very high,with only one or two bases different.Common nucleic acid detection methods have low specificity,and interference sequences with high homology can lead to false positive detection.This study optimized the RCA detection technology and developed a high specific nucleic acid detection technology that combines S1 enzyme,nucleotide derivatives and rolling circle amplification(RCA)to reduce false positives caused by high homology interference with nucleic acids.To provide technical support for realtime detection of micro RNAs of the same family.Research methods: 1.The synthesized 30 bp single strand of nucleic acid was mixed into a complete complementary double strand and a single base mismatch double strand(mismatch is located in the middle of the double strand).Based on the principle of fluorescence resonance energy transfer,the difference in the uncoupling temperature was analyzed.The structure difference of double chain was analyzed by polyacrylamide gel electrophoresis.2.Based on the principle of fluorescence resonance energy transfer,single chain specific nuclease S1 was used to perform enzyme digestion on the complete complementary double chain and single base mismatched double chain,so as to explore the enzyme digestion conditions of S1 enzyme enzyme digestion on the single base mismatched double chain and protect the complete complementary double chain.After enzyme digestion,the sample was electrophoretic and analyzed the structure.3.Construct a double strand formed by the protective probe and the target nucleic acid or interfering nucleic acid,use S1 for enzyme digestion,and use the digested product as primer for RCA reaction,and compare the difference between its specificity and that of the primer without enzyme digestion.4.The 2’F generation was modified in the middle of the synthetic single strand of nucleic acid,and the double strand primer RCA was compared with the unmodified double strand primer RCA.The difference of double strand structure before and after modification was analyzed by polyacrylamide gel electrophoresis.After enzyme digestion,the modified double strand was used as primer for RCA to observe whether the specificity of RCA detection was improved.Results: 1.The uncoupling temperature of 30 bp complete complementary double chain was 75-77 ℃,and that of single base mismatched double chain was 71-73 ℃.Electrophoretic observation showed that there was no difference in the structure of complete complementary double strand and single base mismatch double strand.2.The complete cleavage time of the complete complementary double strand under 1:3diluted S1 enzyme at 50 ℃ was earlier than that of the single base mismatch double strand.It was observed that the mobility of the fully complementary double strand after enzyme digestion was different from that of the single base mismatch double strand,but the difference showed no obvious fluorescence curve.3.The fluorescence value of the complete complementary double strand was higher than that of the single base mismatch double strand when the double strand was digested by 1:3 diluted S1 enzyme at 37 ℃.4.The difference of fluorescence value between the 2’F generation modification and the modification in the middle of the complete complementary double strand is less than that of the single base mismatch double strand.The fluorescence value after modification was slightly higher than that before modification.5.In RCA detection,S1 enzyme cleaved 2’F generation modified complete complementary double strand.Conclusion: The combination of S1 enzyme and RCA detection can improve the specificity of RCA detection.The modification of 2’F generation increases the difference of structural stability between complete complementary double strand and single base mismatch double strand.The combination of S1 enzyme and 2’F generation in RCA can further improve the specificity of detection and reduce the non-specific amplification of interfering sequences with high homology with the sequence to be detected.This method improves the RCA detection technique and makes it have higher application value.It provides technical support for real-time fast detection.