Construction And Clinical Application Of Endometrial Cancer Gene Mutation Detection Technology

Part 1 The verification of the principle of branch migration based PCR for mutant-type target enrichment[Purpose] We presented a branch migration based PCR in which a branch migration Blocker was introduced to selectively reduce the amplification efficiency of the wild-type target and enrich the mutant-type target.[Methods] A branch migration blocker(BM Blocker)sequence is introduced into the PCR process.BM Blocker is complementary to wild-type DNA(WT)and has one mismatch with mutant-type DNA(MT).The primer and blocker would undergo branch migration process when they bind to WT.The probability of PCR replication of WT is 1/(1+n)in which n is the overlap region of BM Blocker and primer while the probability of PCR replication of MT is about 1 as the BM Blocker could not bind tightly to MT with one mismatch.Thus,the MT could be enriched through PCR.[Results]We first showed that the branch migration process between the primer and BM Blocker could impose a negative effect on polymerase elongation.High resolution melting(HRM)analysis showed that the BM PCR could elevate the mutation abundance obviously.[Conclusions] Our data firmly demonstrated that the proposed BM PCR was able to enrich low-abundance point mutations during the amplification process.PART 2 Branch migration based selective PCR combined with the DNA probe for the gene mutation of endometrial cancer[Purpose] Enrichment of low abundance point mutations is vital for bioanalysis and molecular diagnosis.Here we present a branch migration based PCR(BM PCR)combined with DNA probe for the enrichment of low-abundance mutations.[Methods] We then designed and synthesized a fluorescent DNA probe whose sequence was complementary to mutant-type DNA and single-base mismatched to wild-type DNA.As for BM PCR,the overlap region between primer and BM blocker is 4-nt and there leaves 9-nt for the blocker to overlap with the probe considering the gap for polymerase elongation.Thus,the mutation site could be set away from the ends of the blocker and probe and the mismatch recognition ability would hardly be affected.The additional probe would cooperate with BM blocker to further enhance the method's discrimination ability toward MT and WT and combine the enrichment step and subsequent analysis step into one.Actually,the blocker strand herein had two functions: it would compete with primer to enrich the abundance of mutant-type DNA;it would also compete with the probe to enhance the discrimination power.Overall,the combination of three components enables the rapid and sensitive detection of low-abundance point mutations.[Results] Sanger sequencing showed that the BM PCR could elevate the mutation abundance from 5% to ~50%.The fluorescent DNA probe could be coupled within the BM PCR process to further enhance the method's specificity and lower down the detection limit to 0.1%.Genomic DNA extracted from haemocytes or tissue samples of cancer patients was further tested with BM PCR and demonstrated the practicability of BM PCR on real clinical samples.[Conclusions] Overall,compared with other mutation enrichment methods,BM PCR was easy to design,convenient to perform and highly efficient.Researchers only need to optimize the temperature settings of the blocker strand and the whole detection process was merely PCR amplification.We anticipate our established BM PCR to be widely adopted in molecular diagnostics and clinical analysis.PART 3 Branch migration based selective PCR combined with endonuclease ?-based signal amplification system for the gene mutation of endometrial cancer[Purpose] The development of detection methods for low abundance point mutations in blood or tissues is becoming a new direction in the detection of cancer and other gene mutation related diseases in the future.We have developed a new and simple method for detecting low abundance point mutations in tissue samples of cancer patients.[Methods] Based on the BM-PCR method mentioned above,we think that the combination of the endonuclease IV signal amplification system will further improve the sensitivity of mutation gene detection.As for endonuclease IV-assisted target recycling probe/blocker system,a s-Blocker is introduced into the system.We first designed and synthesized a dually-labeled fluorescent DNA probe with an apurinic/apyrimidinic site(AP probe)whose sequence was complementary to the target mutant template and a single-base mismatched to wild template.The sequence of the s-Blocker is complementary to the wild-type DNA and thereby a single-base mismatched to the target mutant DNA.The competitive s-Blocker and AP probe compete to bind with the template and one of them will release from the target at a certain melting temperature.Importantly,we designed s-Blocker with the same sequence as the AP-probe.So the melting temperatures of the s-Blocker/wild-type template duplex and the AP-probe/mutant template duplex are almost the same.Overall,the signal of MT are amplified,thus the detection limit is further lower down.[Results] Sanger sequencing results showed that BM-PCR method can increase the mutation abundance from 5% to 40%,whi ch has a high enrichment efficiency.In addition,the detection limit of PTEN R130Q(389G> A)and PTEN rs121909219 mutations in endometrial cancer tissue was reduced to 0.02% and 0.01%,respectively.[Conclusions] In conclusion,we have established a BM-PCR combined with the Endo IV-assisted target recycling probe/blocker system for detection of low-abundance point mutations.To the best of our knowledge,our method is one of the most sensitive approaches for the detection of low-abundance point mutations in real samples,which demonstrates its clinical practicability.We hope that this method can provide reference for the analysis and diagnosis of gene mutation information of endometrial carcinoma.