Multiplexed Mutation Detection In BRCA Gene Of Ovarian Cancer

Part 1 Research on probe/blocker composition system[Purpose] To analyze the reaction mechanisms of the inverse correlation between sensitivity and specificity of the conventional probe/blocker composition system,develop a new probe structure to break the intrinsic inverse correlation between the sensitivity and specificity of the probe/blocker composition system,and improve the performance of the detection system.[Methods] Analyze the reaction mechanisms of probe/blocker composition system,develop the 4-way SELECT system.Taking clinically significant pathogenic mutations as the target model,on the one hand,explore the correlation between sensitivity and specificity over blocker sequence and concentration at the experiment;on the other hand,construct theoretical models for competitive DNA hybridization,and compare it with the experimental data.[Results] It was the inverse correlation in thermodynamics and the kinetic traps that led to the inverse correlation between sensitivity and specificity of the competitive probe/blocker systems.We thoroughly understand the thermodynamics and kinetics of DNA hybridization,and innovatively developed 4-Way SELECT system that breaks the intrinsic inverse correlation between sensitivity and specificity of the competitive DNA hybridization system,the specificity can be optimized to the theoretical maximum,and the sensitivity can be maintained at a relatively high constant value.[Conclusions] We have thoroughly analyzed the reaction mechanisms of competitive DNA hybridization,and constructed complete theoretical models for them.Guided by the theoretical calculation,we developed the 4-way SELECT system which was able to break the intrinsic inverse correlation between sensitivity and specificity over blocker sequence and concentration.Our proposed probe design approach showed great convenience and robustness in nucleic acid analysis especially for multiplexed or high-throughput mutation detectionPart 2 Construction and optimization of the multiplexed detection system based on 4-way SELECT[Purpose] The 4-way SELECT system has break the intrinsic inverse correlation between sensitivity and specificity over blocker sequence and concentration,which showed great convenience and robustness in multiplexed or high-throughput mutation detection.Based on the 4-way SELECT system,to significantly lower down the expenses of multiplexed detection,and optimize the system to achieve the best performance.[Methods] Design the multiplexed probe structure which use the same fluorescent and quencher strands for different probes that target different sequences can greatly reduce the cost of multiplexed detection.Based on the 4-way SELECT system,we constructed Y-shaped and Star-shaped DNA probe for universal detection of different mutations.Furthermore,construct two forms of signal amplification for multi-sites detection to optimize the system’s performance.One,the fake strand is introduced to build a self-amplification multiplex detection system based on the 4-Way SELECT system.Another,an amplification multiplex detection system which based on the 4-Way SELECT system and endonuclease IV enzyme.Taking clinically significant pathogenic mutations as the target model to explore the detection limit of the target strand.[Results] The uniformed 16-plex Y-shaped 4-way SELECT system was able to identify all the tested mutations with high specificities.The detection limit of the star-shaped fluorescent DNA probe for the low abundance of the target sequence is 5%.The self-amplification multiplexed detection system allowed a small amount of target DNA to recycle in the system through the introduction of fake strands,the method has a detection limit of 0.05% for target strand.The universal probe based on the 4-Way SELECT system and the endonuclease IV simplified the universal mode,and the signal amplification effect of endonuclease IV can amplify the signal difference between the mutant-type and wild-type strands,the detection limit is 0.05%.[Conclusions] Based on 4-way SELECT system,we constructed a variety of universal probe structures which greatly reduces expenses of detection,and the performance were all wonderful.Among them,the self-amplification multiplexed detection system and the universal probe based on the 4-Way SELECT system and the endonuclease IV had a detection limit of 0.05% for the target strands,which greatly improves the performance of universal probes.Part 3 Detection of clinical samples[Purpose] To prove the clinical validation of four types of universal probes based on 4-Way SELECT system.Use the universal probes based on 4-Way SELECT system to detect multiple germline pathogenic mutation sites in ovarian cancer.[Methods] We obtained genomic DNAs extracted from the blood samples of ovarian cancer patients and healthy volunteers.On the one hand,using sanger sequence method and next-generation sequencing to detect the genomic DNA of the sample.On the other hand,using the universal probes based on 4-Way SELECT system to detect the genomic DNA of the sample.Compare the experimental results with the results of Sanger sequencing and next-generation sequencing to verify the feasibility of the method.[Results]The star-shaped universal probe system based on the separation mode 4-Way SELECT and the Y-shaped universal system based on the non-separation mode 4-Way SELECT can only distinguish between mutant and wild-type DNA for the actual sample.The self-amplification multiplexed detection system and the universal probe based on the 4-Way SELECT system and the endonuclease IV had a detection limit of 0.05% of the real clinical samples.The results provided by the 4-way SELECT system were in accordance with the Sanger sequencing results and next-generation sequencing,demonstrating the practicability of our system in real clinical applications.[Conclusions] This section built the basic flow of real clinical sample detection and proves the clinical practicability of four different mode probes based on 4-Way SELECT system.Among them,the self-amplification multiplexed detection system and the universal probe based on the 4-Way SELECT system and the endonuclease IV are effective,and the methods can provide a diagnostic reference for the information of multiple mutations of ovarian cancer BRCA gene.