Analysis Of Single Base Mutation And 5-hydroxymethyl Modification Based On The Mechanism Of Nucleic Acid Ligation Amplification

Most genetic materials of organisms are DNA,through the transcription and translation processes,the hereditary information stored in base sequence is transmitted to proteins,and then involves in the regulation of life activities.Genetic mutations and DNA epigenetic modifications are going to influence the regular development of origanisms.There are close relationships between some of human diseases and gene expressing.Therefore,to detect mutant gene and epigenetic modification has significant meaning in life science and pathobiology.Based on the ability of ligase to recognize single base mismatch,through nucleotide amplification,such as polymerase chain reaction and loop-mediated isothermal amplification,we accomplished the analysis of single base mutant and 5-hydroxymethyl cytosine.This approach offers a pathway for the research on molecular mechanism and biomedical diagnosis.With the single base mutation on DNA,the function of protein has changed,which will influent the regular development of organisms.Therefore,it is very necessary to build a method for detection of single base mutation.By mutant-type KRAS gene as target,we established an approach to detect the SNP locus on KRAS gene,which combined the cycle ligation reaction and PCR technology.By using this method,we can detect the mutant-type gene even the concentration of it was 10 aM,but the sensitivity and specificity of this method were still needed to be improved.DNA epigenetic modification is mainly refers to the modification on 5'-C of cytosine,including methylation and hydroxymethylation.The products of this process are 5-methylcytosine(5-mC)and 5-hydroxymethtlcytosine(5-hmC),respectively.Demethylation process is an automatic regulation occurred in vivo,which regulated the development of cells.As a biomarker in demethylation process,5-hmC also plays an important role in other basic processes of life.Thus,it is an urgent problem needed to be solved that how to establish a sensitive and specific method for detection of 5-hmC.In this paper,in order to solve the problem that 5-hmC is very difficult to distinguish in the presence of 5-mC and C,by ligation-digestion-LAMP detection system,we established a sensitive and specific method for detection of 5-hmC,which integrated Hpa? enzyme digestion,oxidation and treatment using potassium perruthenate and sodium bisulfate,respectively,that converted the distinction of 5-hmC into that of single base differences.The detection limit of this method for 5-hmC was as low as 100 aM,and there was a liner relationship between-logC5-hmC and POI of fluorescent curves during the concentration range between 100 aM to 1 nM.Meanwhile,if the concentration of 5-mC and C is lower than 10 fM,there is no effect during the detection process of 5-hmC.In the sample contained 5-hmC,5-mC and C,the total concentration of which is 10 fM,we successfully detected the corresponding fluorescent signal of 5-hmC only.Finally,we used this method to detect the amount of 5-hmC in sample of mouse brain.