Construction And Application Of Multiple Mutation Detection System For Gynecological Tumor Pathogenic Genes

Part 1 Research on improving DNA probe discrimination[Purpose] The non-specific cleavage activity of nuclease seriously affects the detection performance of DNA probes.By qualitatively and quantitatively solving the problem of nuclease non-specific cleavage activity in the auxiliary enzyme/probe detection system,and creating a new guide chain(guiding)auxiliary enzyme/probe detection system,so as to improve the discrimination of DNA probes.Finally,it provides a new method for realizing low-abundance gene mutation detection.[Methods] Through analyzing the binding preference of endonuclease IV(Endo IV),apurinic and apyrimidinic endonuclease(APE-1)and lambda exonuclease,adding double-stranded DNA(ds DNA)to inhibit the self-cleavage activity of on the probe by Endo IV,APE-1 and lambda exonuclease,a new guiding strand co-enzyme/probe detection system was designed.And pathogenic mutations with clear clinical significance were set as the target model,in the experimental aspect,whether the binding preference of Endo IV,APE-1 and lambda exonuclease with ds DNA change along with the change of ds DNA length and concentration was explored;in the theoretical aspect,mathematical model was constructed in order to accurately predict and regulate the cleavage rate of nucleases,which is verified by experimental results.[Results] We have found a way to inhibit the self-cleavage of nucleases: adding irrelevant ds DNA to the detection system immediately inhibits self-cleavage;constructing mathematical models through experimental data,so that the distribution of nucleases in the system can be precisely regulated;through the establishment of a new guiding strand auxiliary enzyme/probe detection system and the detection limit of this method to distinguish between wild strand and mutant strand is 0.01%.[Conclusions] The root cause of the non-specific cleavage activity of the nuclease in the auxiliary enzyme/probe detection system is that the nuclease has a certain binding ability to single-stranded DNA(ss DNA);adding ds DNA to the auxiliary enzyme/probe detection system can inhibit the self-cleavage of the single-stranded nucleic acid probe by the nuclease in the detection system and meanwhile enables detection system to maintain a high detection efficiency for mutant strand.Part 2 Construction and optimization of a 2-way DNA cascade reaction multiple mutation detection system[Purpose] To establish a stable and common detection system that can be used for low-abundance mutation detection,and reduce the cost of DNA fluorescent probes for multiple mutation detection.And to optimize the public detection system to make the performance of the public DNA fluorescent probe detection system reach the best.[Methods] Through constructing a 2-way interlocking DNA cascade reaction/auxiliary enzyme detection system,a common multiple mutation detection system was designed.The same probes labeled with fluorescent groups and quenching groups can be used to perform detection on different targets,which greatly reduce the cost of detecting multiple mutation sites with DNA probes.The 2-way interlocking DNA cascade reaction/auxiliary enzyme detection system stably realized the common multiple mutation detection system through the guide strand and the bridge strand.With the assistance of the nucleic acid tools Endo IV and lambda exonuclease,it forms circular amplification effect,so as to realize the distinction between mutant strand and wild strand;Genomic DNA was obtained from tissue samples of healthy people and endometrial cancer patients,on the one hand,sanger sequencing was used to detect the genomic DNA of the sample to determine the mutation abundance of the genomic DNA of the sample;On the other hand,the auxiliary enzyme/2-way interlocking DNA cascade reaction detection system was used to detect genomic DNA.Finally,the experimental results were compared with the sanger sequencing results to verify the practical feasibility of the method.[Results] The universal detection system of 2-way DNA cascade reaction assisted by Endo IV detected three hotspot mutations under uniform zero optimization conditions.The detection limit of this method was 0.1% for the target strand;in addition,the 2-way DNA cascade reaction common detection system assisted by lambda exonuclease has also a detection limit of 0.1% for the three hotspot mutations.What’s more,we also established a prediction model to predict the application effect of the 2-way DNA cascade reaction common detection system.Finally,through detecting the PTEN gene mutation in clinical specimens of endometrial cancer,the2-way DNA cascade reaction common detection system was proved to be feasible.[Conclusions] Based on the 2-way DNA cascade reaction system,a public detection system can be constructed,and the detection effect is good,which greatly reduces the cost of DNA fluorescent probes for multiple detection,simplifies the operation process,and saves detection time.Among them,the 2-way DNA cascade reaction common detection system has a detection limit of 0.1% for the target strand,which greatly improves the efficiency of the common probe.Part 3 Construction and optimization of multiple mutation detection system based on probe melting curve method[Purpose] To establish a common probe melting curve DNA cascade reaction multiple mutation detection system that can be used for low-abundance mutation detection to reduce the cost of multiple mutation detection;to optimize the common detection system of the probe melting curve DNA cascade reaction to make the performance of the common probes melting curve DNA cascade reaction detection system reach the best;to establish a mathematical model to simulate the experiment of the probe melting curve DNA cascade reaction common detection system,while the effect is predicted.[Methods] Through constructing a DNA cascade reaction mutation detection system based on the melting curve of the probe,a public multiple mutation detection system was designed.The same probe labeled with a fluorescent group(FAM)and a quenching group(BHQ)can be used for the detection of different target mutation points,which greatly reduces the cost of detecting multiple mutation points.The detection of multiple mutations was realized by the probe melting curve DNA cascade reaction mutation detection system through a bridge strand.As the melting curve test proceeds,the DNA cascade reaction gradually occurs,causing changes in the secondary structure of the DNA probe,so as to achieve the distinguishing effect.A mathematical model was also established to predict the experimental results to screen the best reaction conditions.Finally,genomic DNA was obtained from tissue samples of healthy people and ovarian cancer patients.On the one hand,the genomic DNA of the sample was sequenced by sanger to determine the sample genome DNA mutation abundance.On the other hand,the probe melting curve DNA cascade reaction mutation detection system was used to detect genomic DNA.Finally,the experimental results were compared with the sanger sequencing results to verify the practical feasibility of the method.[Results] The probe melting curve DNA cascade reaction mutation detection system detects 6 hotspot mutations on the BRCA gene under uniform zero-optimized conditions,and all have good discrimination.Compared with the effect of traditional melting curve technology,the distinguishing effect is equivalent;this method can accurately reflect the experimental results through the established mathematical model,thereby predicting the optimal reaction conditions of the experiment;finally,detecting BRCA gene mutations in clinical specimens of ovarian cancer,verified the clinical feasibility of the probe melting curve DNA cascade reaction common detection system.[Conclusions] Based on the probe melting curve interlocking DNA cascade reaction detection system,a public detection system can be constructed,and the detection effect is good,which reduces the cost of multiplexed mutation detection,simplifies the operation process,and saves the detection time.The successful detection in actual samples indicates that the system is clinically popular.