Designed Mismatched DNA Improved The DNA Ligase Reaction Based On Detection Of Single Nucleotide Variation

The most common form of genetic variation is single-nucleotide polymorphism(SNP),which is closely related to human diseases and drug efficiency.SNP detection has been considered as a promising tool for early diagnosis of malignancy risk.A comprehensive understanding of SNP can provide valuable information and be used to guide clinical diagnosis and individual drugs.Therefore,simple and accurate SNP detection is very important.Due to the low commercial cost and the flexibility of nucleic acid substrate identification,the T4 DNA ligase is widely used in biomolecular engineering,especially for specific nucleic acid sequence detection.This paper explores the effect of the introduction of additional mismatching in the identification of single base mutant sites on the target gene fragment with different ligase mediated connections based on T4 DNA ligase.In this paper,the interlocking activity of different DNA ligase was systematically verified by the padlock probe reaction,and the effects of introducing extra mismatched ligase on the specificity of ligase were analyzed.The results showed that the additional base mismatch enhanced the specificity of T4 DNA ligase for nucleic acid with single base variation.Detection of single base DNA mutation revealed that the additional mismatch in the adjacent positions of the junction points could significantly enhance the specificity of T4 DNA ligase.Among them,the most effective mismatch base pairs can increase the specificity of T4 DNA ligase by more than 600 times.For the detection of single base RNA mutation,it was found that the optimal location of additional mismatch was different for different bases,and the sensitivity of T4 DNA ligase to DNA substrate was higher than that of RNA/DNA compound.In addition,the coupling reaction mediated by Taq DNA ligase was also analyzed,and the experimental results showed that the additional mismatch had little effect on the specificity of Taq DNA ligase on substrate recognition.This paper studies the ligation efficiency of different ligase and the identification specificity of different substrates.It is confirmed that the additional mismatch can improve the specificity of T4 DNA ligase for substrate identification and improve the accuracy of single base mutation detection.The conclusions are useful for the detection of specific nucleic acid loci with lock probe as a model,and it can also promote the development of clinical diagnostic techniques and individualized drugs.