Detection Of Mutations In Single-stranded DNA Molecules Using Surface-enhanced Raman Spectroscop

DNA is the main genetic material of life,guiding biological development and the operation of life functions.The parent transmits part of his DNA replication to the offspring to complete the dissemination and expression of the offspring’s traits.In the process of inheritance,genes can faithfully maintain the basic characteristics of their parent organisms.Under the influence of heredity and environment,they can also produce "mutations" and "variations" in the case of reproduction of offspring.The vast majority of mutations lead to genetic diseases that the parent does not have,which poses a certain threat to the development of human life.There are many ways to detect DNA mutations.Surface-enhanced Raman spectroscopy(SERS)analysis technology has the advantages of simplicity,non-destructiveness,no fluorescence interference,time-consuming,trace amount,etc.It can obtain the unique fingerprint information of nucleic acid and is an effective means to detect DNA mutations.However,there are still some difficulties in SERS detection of DNA mutations.In response to these difficulties,this paper designs a new method to grasp the effect of base stacking inside DNA,and realize the analysis of mutations and mutation sites of single-stranded DNA molecules,including the following two contents:1.Detection of A/G mutations in ssDNA based on Ag@ITNPs label-free SERSIt is generally believed that the self-folding of single-stranded DNA(ssDNA)depends on the hydrophobic effect of its internal bases,but the folding of single-stranded DNA in solution is not completely random,but will be affected by the stacking effect of adjacent bases.In this work,we developed a new method to explore the stacking regularity between adjacent bases using SERS.Acidic titanium ions were introduced into iodide-encapsulated silver nanoparticles(Ag@ITNPs)as aggregating agents,and symmetric spectra were obtained by normalizing the peak intensity of deoxyribose at 955 cm-1.Based on the influence of adjacent base stacking on the spectrum,the SERS method was used to accurately identify A/G point mutations and mutation sites.In addition,the base content and DNA frameshift mutations of ssDNA were precisely analyzed.The experimental process of this method is simple,and it can accurately capture the changes of the base ring respiration peak intensity caused by different adjacent bases,which will provide potential application value in the field of gene diagnosis.2.Detection of C/T mutations in ssDNA based on label-free SERS of Ag@SCNPsRecent developments in molecular spectroscopy have widened the scope of SERS for detection of nucleic acids.In order to solve the interference of impurity signals in SERS analysis that hamper the reliable detection of DNA,Ag nanoparticles modified with thiosulfate ions were used to obtain SERS signals of DNA molecules in aqueous solutions,which showed good reproducibility.By using thiosulfate ions and calcium ions,this method not only eliminated the influence of citrate on DNA signals completely but also obtained the signals for all bases indiscriminately,including the T base that was considered to have low Raman activity.Subsequently,the base stacking rule was used to identify mutations arising from C/T transition.It further identified the mutation sites of single-base C/T transition using this platform for the first time.This method has wide application prospects in DNA analysis,DNA sequencing,and genetic testin.