Construction Of New Enrichment And Detection Technology For Detecting Single-gene Mutation Diseases By Plasma DNA

Part 1 Research on the Enrichment of Cell-free DNA Based on SSB-PCR[Purpose] To analyze the difficulties in the detection of ct DNA and cffDNA and find out the differences between ct DNA,cffDNA and cf DNA.Construct a method for enriching short-strand DNA.Analyze the feasibility and univer Sality of the principle of the method.To explore the Application of the enrichment method in the detection of low abundance of ct DNA and the Application of non-invasive prenatal testing of single-gene hereditary diseases.[Methods] Consult the literature to analyze the detection difficulty of ct DNA,cffDNA and the difference in properties with cf DNA.Design SSB-PCR enrichment system and construct theoretical model.In the aspect of experiment,the differences in the enrichment of long-strand and short-strand DNA between SSB-PCR and traditional PCR mearsure by Sanger sequencing.Amplifylow-abundance short-stranded DNA by SSB-PCR and traditional PCR,and evaluate the enrichment effect of SSB-PCR on low-abundance ct DNA.Take the non-invasive prenatal diagnosis of single-gene genetic disease as an example,the ratio of long strand to short strand was 9:1,and the mutation abundance is 45%,50%,55%in the mxture.Verify the enrichment effect by SSB-PCR and traditional PCR.Establish the random break model of DNA,and design interference DNA with different lengths.Amplify the low concentration mixtue through SSB-PCR and traditional PCR with the presence of interference strands,and compare the enrichment effect by Sanger sequencing.[Results] Based on the single base mutation IVS-2-654 in β-tha Lassemia,we constructed a SSB-PCR system,the blockers and Primers were designed under the guidance of the theoretical model.The model and experiments proved the feasibility and universality of the principle.SSB-PCR enrichment system can specifically enrich short DNA,with low abundance and the enrichment abundance is as low as 2%.Under the concentration of 40 p M,the Sanger sequencing results showed that SSB-PCR had a clear distinction between the original mutation abundance of 45%,50% and 55% of DNA mixture.Under the guidance of the random fracture model,the Sanger sequencing results show that SSB-PCR can enrich 0.4 fm DNA mixture,and effectively distinguish the three mutations.[Conclusions] We analyzed the length difference of ct DNA,cffDNA,cf DNA and constructed a universal SSB-PCR enrichment system.Theoretical model calculation and experimental results have proved that SSB-PCR has a good effect of enriching short DNA.SSB-PCR can effectively enrich the abundance of short DNA,and has the potential in ct DNA.In the diagnosis of single base mutation IVS-II-654 of β-tha Lassemia,it can effectively distinguish wild homozygous short DNA,heterozygote short DNA,mutation homozygous short DNA,which provides a reference for the applicatioin of NIPT in single-gene genetic diseases.Part 2 Research on Self-Amplifying Probe Detection System Based on Endonuclease IV[Purpose] To analyze the difficulties of the method of Probe detection in ct DNA and analyze the shortcomings of ct DNA detection method with endonuclease IV in present.Design a new ct DNA detection system though nucleic acid probe and analyze the feasibility and universality of the method.Explore the performance of the new method in the detection of ct DNA at low mutation abundance and realize applicatioin in ct DNA.[Methods] Consult the literature to analyze the advantages and disadvantages of the method,and summarize the characteristics of endonuclease IV.Design the self-amplification probe detection system and construct the theoretical model.In the aspect of experiment,verify the feasibility of principle by comparing the rise slope of fluorescence signal between wild-type DNA and mutant-type DNA.Verify the universality of the principle with six types of mismatches.According to the low abundance of the mixture of wild-type DNA and mutant-type DNA,explore the detection limit.Amplify the genomic DNA by the asymmetric PCR,and detect the post-PCR product to explore the feasibility of clinical application.[Results] The self-amplifying probe method can effectively distinguish wild-type DNA from mutant-type DNA,with good effective in 6 kinds of mismatches.Take EGFRL858 R and PTENR130 Q as examples,the detection limit of synthetic DNA is as low as 0.02%.In the application of clinical sample,the method can detect point mutation at the abundance of 0.05%.[Conclusions] We analyzed the requirements of probe detection method,compared and summarized the features of current probe detection methodes with endonuclease IV,and added strand migration system to previous system.According to the theoretical model calculation and experimental results,it is proved that the self-amplifying probe method is feasible,universal and has low detection limit.And the detection temperature is constant,which greatly reduces the optimization cost in different sequence detection.Part3 Research on SIR Probe Detection System Based on Strand Migration System[Purpose] To analyze the core cause of the difficulty in quantitative the abundance of mutaion of probe based method and design a new probe system and then analyze the feasibility of its principle.Explore the performance of a new probe method in the detection of low abundance mutations in ct DNA and its application in non-invasive prenatal testing in single gene genetic diseases.[Methods] Analyze the quantitative detection mode of probe and design SIR Probe system creatively.Taking single-base point mutation as an example,construct a strand migration thermodynamic model.In the aspect of experiment,explore the relationship between the ratio of Probe-T and Probe-R.Detect single-base point mutation KRASG13 D to verify the feasibility of the principle.Mix WT and MT into different abundances to explore the lowest quantitative detection of mutation abundance.Synthesize the long DNA strands of the KRASG13 D and mix the WT and MT into different abundance and ss DNA will be amplified by asymmetric PCR.Detected the post-PCR product by SIR Probe system.Design SIR system for detecting IVS-2-654.Simulate the NIPT inwhich the mutation abundance was 45%,50% and 55% respectively,and then detect them under different concentrations.Synthesize the long-stranded IVS-2-654 DNA sequence to simulate the detection of the clinical sample and detect after asymmetric PCR.Combined with SSB-PCR and SIR system,the products were detected with SIR and Sanger sequencing..[Results] The fundamental reason for the difficulty of quantitative detection of nucleic acid probe is that it depends on the relationship between signal and substrate concentration,there is no reference,and it is interfered by irrelevant DNA strands,while the SIR Probe system innovatively designed.Construct its own sequence as a reference and is not disturbed by irrelevant strands.The SIR system can effectively distinguish between wild-type DNA and mutant-type DNA,and can quantitatively detect abundance at different concentrations in KRASG13 D.The detection limit of SIR system in synthetic DNA KRASG13 D is 2%.In the simulated real sample detection,the SIR system can still achieve constant detection of different concentrations with the same abundance.And the detection limit is 2%.The SIR system can effectively distinguish three different mutation abundances of single base mutation IVS-II-654 in β-tha Lassemia for non-invasive prenatal diagnosis,and show a stable detection at different concentrations.45%,50% and 55% can be effectively distinguished in synthetic DNA detection and post-PCR detection.Combined with SSB-PCR,the SIR system can detect lower concentration of short-strand DNA,and significantly distinguish three different mutation abundances of non-invasive prenatal diagnosis,thus reducing the difficulty of detection.[Conclusions] We deeply studied the reaction principle of strand migration and constructed the theoretical model of strand migration SIR system.According to the theoretical model calculation,we creatively designed the SIR system,abandoning the correlation between the concentration and the strandard curve of nucleic acid probe quantitative detection.The detection system based on SIR system can be applied to the detection of single base mutation,and the detection effect is well,and the detection limit is as low as 2%.The SIR system has a broad application prospect in the quantitative detection of single base mutation abundance.The results of IVS-2-654 detection of β-thalassemia with single base mutation suggest the possibility of using SIR system in non-invasive prenatal testing.Among them,the detection results combined SSB-PCR provide experimental proof for the subsequent development of SIR system for non-invasive prenatal testing.