A New Method Research For The Detection Of KRAS Gene Mutation In Colorectal Cancer By Ligation Reaction And Loop-mediated Isothermal Amplification

Objective: This paper intends to construct a new amplification method based on ligation reaction and loop-mediated isothermal amplification(LAMP)for the detection of KRAS mutation with high sensitivity and specificity.Methods: In this work,the feasibility of the ligation-initiated loop-mediated isothermal amplification method was first verified.Mutant KRAS DNA(mut DNA)can be paired with SLS1 and SLS2 as ligation substrates.In the presence of ligase,the dumbbell-shaped structure(DSS)formed by the specific ligation of two substrates(SLS1 and SLS2)and then acts as a template to initiate the LAMP amplification reaction,resulting in a large number of double strand DNA(ds DNA).As a result,a significant fluorescence signal is achieved by the preferential binding between the fluorescence dye SYBR Green I with ds DNA.While,compared with the mut DNA,there is one more position mismatch between wt DNA and SLS2.Thus,no ligation occurs and without the formation of DSS in the presence of wt DNA,there is a negligible fluorescence background from the detection of wt DNA.After the feasibility analysis,the mismatch site,the ligation temperature,the numbers of ligation cycles,the Bst DNA polymerase concentration and the LAMP temperature in the experiment are optimized,and then the linear range and specificity of the scheme are investigated under optimal experimental conditions.Finally,this method has been applied for the detection of mutation from the tissue samples of colorectal cancer,and the results obtained are compared with those of PCR.Results: This work has successfully developed a ligation-initiated LAMP method for the detection of KRAS mutation.Under the optimal experimental conditions(ligation temperature: 63 °C,cycle number: 30 cycles,the concentration of Bst DNA polymerase: 0.4 U/?L,LAMP temperature: 65 °C),This ligation-initiated LAMP method not only achieves an ultrahigh sensitivity and specificity for detection of mut DNA at a M level but also can quantitate the rare mutation even in a large excess of coexisting wt DNAwith a selectivity factor down to 0.1%.This method has been successfully applied to the accurate detection of mutation from the tissue samples of patients with colorectal cancer,which is in good agreement with the results of PCR.Conclusion: The ligation-initiated LAMP method holds the great potential application for the detection of DNA mutation,and also has a promising prospect in disease diagnostics,personalized medicine and biomedical research.Presenting a promising tool for clinical diagnosis and treatment.