A Method Of Gene Analysis Based On Rolling Circle Amplification

Genetic defects and mutations are the source of genetic diseases and many chronic diseases that are difficult to cure.So the acquisition of genetic information has become the cornerstone of precision medicine.Modern medicine has shown that the specific genetic variation that occurs in long-term specific living conditions is the main cause of cancer.Therefore,the detection and analysis of genetic information has become an important tool for biomedical research and clinical diagnosis.In this study,we mainly explored the techniques that are the analysis and detection methods of gene mutation related to cancer,especially single nucleotide variation.Through combining the design of nucleic acid probe with rolling circle amplification(RCA)technique,the high specificity and sensitivity detection of single nucleotide variation was realized at room temperature.The purpose of this paper is to explore the gene detection methods that are high selectivity for single nucleotide variation.Here we use the technique that combines padlocks with rolling circle amplification so that it can be applied to a wide range of samples and are capable of rapid detection at room temperature.The specific processes involve forming a circular DNA with a specific sequence by identifying the target gene sequence and signal amplification by RCA reaction.Thermodynamics was successfully introduced into the design of probe in order to improve the ability of the selectivity for single nucleotide variation.Compared with padlock method,the specificity of the probe is improved by nearly 10 times.In addition,the rapid amplification of signal was carried out by RCA reaction.By optimizing the related reaction factors such as DNA polymerase,temperature and so on,it was verified that the controlled linear and exponential RCA reaction were achieved.Finally the T790 M mutation loci of EGFR,which is the core single nucleotide mutation of lung cancer,was used as the model to realize the high selectivity of single nucleotide variation.Thus the feasibility of this technique was verified.Based on the research of this study,we developed the detection method that combined probe which meets thermodynamics balance with RCA reaction to realize the high specificity for trace the gene variant at room temperature.