Minisequencing-Application Of A New Detection Method In RAS Mutation Detection

Colorectal cancer is a common malignant tumor that causes serious harm to human health.Epidermal growth factor receptor(EGFR)targeted drugs can significantly improve the survival of patients with colorectal cancer.However,recent studies have shown that EGFR targeted drug therapy is not effective for patients who harbor RAS mutations.In addition,mutation types may cause their unique biological effects,suggesting that it makes sense to detect and identify the types of RAS mutations.In this thesis,combined with the MeltArray technology and LNA modification technology,we proposed a novel mutation detection method-minisequencing,and applied it to the detection and differentiation of RAS mutations.The first chapter gives a brief review regarding the relationship between RAS mutations and molecular targeted therapy of colorectal cancer,the common detection methods of RAS mutation and the common types of tumor specimens.This is followed by an introduction of the clinical status on detection for RAS mutations,the lock nucleic acid,and the propose of this thesis.The second chapter illustrates the principle of MeltArray technology,and describes the idea of applying MeltArray technology to minisequencing.The establishment of MeltArray minisequencing system includes selection of detection sites,experimental design of the system,construction of the plasmids,design of the primers/probes,and establishment of simplex and multiplex system as well.In the third chapter,by using second site of KRAS codon 12 as a model target,we investigated the feasibility of applying MeltArray technology to minisequencing.Based on the results,we established a 12-site RAS MeltArray minisequencing system,which could detect and distinguish 24 common RAS mutation types in three reaction tubes.The sensitivity of the system was 30 copies/reaction,and the selectivity was 5%-10%.We detected 167 clinical colorectal cancer specimens,from which 54 positive specimens(54/167,32.3%)were detected.Compared with Sanger sequencing,seven more positive specimens were detected.All the inconsistent results were subject to an allele-specific PCR assay,and the results were fully concordance with our mimisequenciNg metheod.In summary,We established a new detection method that can distinguish multiple mutation types at the same site.This method proved to be suitable for RAS mutation type identification.The method proposed in this paper is expected to be useful for other somatic mutations and could help patients achieve more precise personalized treatment.