Enrichment And Detection Of Rare DNA Mutations Based On Blocker-induced Hyperbranched Rolling Circle Amplification

Gene mutations are important biomarkers for cancer diagnosis and surveillance.Sensitive identification of single nucleotide mutations can help achieve precision medicine.Existing detection methods include sequencing,probe hybridization,amplification reaction based on primer extension,enzyme digestion technology based on enzyme specific recognition,and ligation reaction.There are many deficiencies,such as the need for thermal cycling,expensive instruments,and accurate Temperature control,professional technicians,insufficient sensitivity and specificity,etc.In response to the above problems,this paper innovatively proposes a new type of sensitivity,simplicity,low cost and high specificity based on blocker displacement amplification system(BDA)and hyperbranched rolling circle amplification(HRCA)technology.The single-nucleotide mutation isothermal amplification and detection method,the main research contents are as follows:First,the detection principle is proposed and the feasibility of the method is experimentally verified.The conditions for amplification temperature,amplification time,primer 2 and dNTP concentration are optimized,and the performance of the method for detecting DNA single base mutations is measured through experiments.The results show that after adding a blocker that exactly matches the wild type in the mutant and wild-type genes,the 3 'end of the padlock probe can hybridize reasonably with the blocker,resulting in a large number of padlock probes The mutant target hybridizes and extends,connecting in a "gap-fill" form to form a loop,and only a small amount of wild-type corresponding padlock probes can form a loop;then a long ssDNA product is amplified under the action of primer 1,Primer 2 and the blocking agent continue to hybridize on this long chain,and finally achieve selective isothermal amplification of mutant targets.A known or unknown single nucleotide variation with an allele frequency of 0.1% can be detected.This method theoretically overcomes the temperature sensitivity of conventional thermodynamic detection of single-base differential nucleic acids,solves the shortcomings of PCR that requires thermal cycling and professional technicians,and is expensive.It is a low-abundance unknown or known single-base variation detection Provides new directions.Secondly,the experiment proves that the method can be applied to the amplification of cluster cancer mutations,and can detect 0.1% KRAS single base variation in genomic DNA extracted from cells.The experimental results are perfectly verified by Sanger sequencing.It is of great significance to promote the development of in vitro diagnosis and precise clinical treatment,and provides a new basis for the diagnosis of point-of-care in resource-poor areas.Finally,the principle and method of in situ detection of mRNA expressed by KRAS mutations in cells were proposed.Direct detection of dsDNA was designed,and preliminary in situ imaging effect of mRNA was obtained,which provided a new idea for cell in situ detection.Due to different local environments,cell heterogeneity and migration,this method provides single-molecule-level sensitivity and ultra-high specificity,and is expected to promote the development of related biomedical research using mRNA mutations as biomarkers for diagnosis.