Highly Sensitive Analysis Of MicroRNA And Gene Mutation Based On Novel Nucleic Acid Amplification Technology

The transcription of genomic DNA into RNA and the translation of RNA into proteins is the basic process of life activities,which plays a vital role in cell growth metabolism and apoptosis.As the basic genetic material regulating physiological activities,nucleic acids carry all the genetic information in life processes and plays a critical role in the major life phenomena such as growth,development,inheritance and variation of all organisms.MicroRNA is a kind of endogenous,non-coding small RNA with relatively short length.The microRNA plays an important role in regulating gene expression,cell proliferation,differentiation and apoptosis by specifically binding to target messenger RNA(mRNA)to inhibit protein translation or induce mRNA degradation.Abnormal expression of microRNA is also closely related to many diseases,especially the development of tumor.In addition,gene mutations caused by single nucleotide polymorphisms(SNPs)can usually cause certain phenotypic changes.The SNPs assay is of great significance in research of pathogenic mechanism,gene function,and personalized medicine.The mRNA mutations at specific sites directly determine the function of the translation protein,which is closely related to the development of various diseases,including cancer.The tumor-associated mRNA mutations of genetic mutation have recently attracted much attention,which can act as specific biomarkers for evaluating the migration of tumor cells in vivo or blood.Therefore,the microRNA and gene mutation assays are significant in gene function and genetic disease research,disease diagnosis,especially the early diagnosis and treatment of cancer.In view of the above important nucleic acid biomarkers,this paper develops a series of simple,sensitive and specific quantitative detection methods for microRNA and gene mutation through innovative design of novel and feasible new nucleic acid amplification technology.(1)A novel and sensitive microRNA assay is developed based on a target triggered loop-mediated isothermal amplification(TT-LAMP)mechanism.In this study,a stem-loop template DNA(SLTD)and a stem-loop primer(SLP)are rationally designed.Only with the SLTD and the SLP,a double stem-loop DNA,the starting material of subsequent LAMP,can be directly formed in a one-step reaction in the presence of microRNA.This new approach remarkably simplifies the probe design,shortens the length of the template,and enhances the generation efficiency of the double stem-loop DNAs,which greatly improves the detection sensitivity of TT-LAMP.Only with one-type of DNA polymerase in the one-step operation,as low as 100 aM(1 zmol)target microRNA can be unequivocally detected,and the single nucleotide discrimination is also accomplished.The sensitive and accurate detection of microRNA is of great significance for the study of its biological functions and the application of biomedicine.(2)Although TT-LAMP method has achieved good results,it is still limited in the analysis of double-stranded DNA and the differentiation of single-base differences.We developed a feasible LAMP amplification based on ligation reaction to achieve high selectivity analysis of SNPs.The ligase-mediated ligation reaction has excellent identification characteristics for single base mutations.The ligation reaction is combined with LAMP amplification,in which two DNA probes are skillfully designed containing the target-specific recognition sequence and the stem-loop structure for LAMP amplification.The probes can be ligated to generate a double stem-loop DNA structure in the present of mutDNA.The subsequent LAMP amplification is performed with the ligation products as the starting material for cycling amplification.The ligation reaction and LAMP amplification are not proceed with the template of wtDNA due to the mismatched base at the terminal.The high sensitivity and specificity is achieved with the proposed ligation-based LAMP that as low as 10 aM mutDNA can be distinctly detected under the isothermal condition,and 0.1%mutated DNA can be clearly discerned in a high background of wild-type sequences.On this basis,we further introduced additional mismatched bases at the third base of the 3'end of the probe.When the end base of the probe is matched with the mutDNA,the ligation reaction can be normally performed and the mismatched base will not affect the sensitivity.Nonetheless,the non-specific amplification can be further reduced effectively due to the mismatch with the 3' terminal base of the probe and the existence of the artificial mismatched base by the template of wtDNA.The high sensitivity is maintained based on the ligation-LAMP with modified probe(as low as 10 aM mutDNA can be detected),meanwhile,excellent specificity is also achieved which allows the clearly discrimination of as low as 0.01%mutDNA target in a high background of wtDNA,which has important significance for the study of gene function and the diagnosis of diseases,especially the early diagnosis of cancer.(3)In recent years,tumor-associated mRNA mutations have been highly valued as the specific biomarkers for evaluating the migration of tumor cells in organisms or blood.Simultaneously,the precise determination of mutant mRNA in a single cell is also of great significance for further revealing the complexity and heterogeneity of cancer development.All of them require highly selective and precise quantitative detection of mutant mRNA.By integrating the outstanding features of a rationally designed peptide nucleic acid(PNA)clamp for highly selective discrimination of single nucleotide variation,and droplet digital PCR for ultrasensitive and precise quantification,an ultra-sensitive and highly specific droplet digital reverse transcription PCR(ddRT-PCR)method is developed for mutant mRNA analysis.This ddRT-PCR assay exhibits ultrahigh sensitivity,which allows the accurate detection of as low as 1 aM mutant mRNA target at the single cell and single molecule level.Meanwhile,excellent specificity is also achieved which allows the clearly discrimination of as low as 0.01%mutant mRNA target in a high background of wild-type mRNA.This robust PNA clamp-based ddRT-PCR assay may serve as a powerful tool for the accurate and precise determination of extremely low copy numbers of mutant mRNA in single cells or in complex blood samples,which holds great promise for the study of cellular heterogeneity at the single cell level,and for the liquid biopsy-related biomedical research by using mRNA mutation as the biomarker.