Nucleic Acid Amplification Method Based On Restriction Effect And Its Application

In recent years,transcriptome sequencing techniques at the single-cell level have become extremely important research tools in the field of molecular biology.These techniques enable effective transcriptome studies at the single cell level and can uncover high-resolution cell type diversity,as well as help to fully understand the interactions of various cell types in complex heterogeneous tissues.However,the amount of total RNA in a single cell is too little to generate expression profiles from individual cells which still remains challenging.Current available library building protocols usually require pre-amplification before subsequent library building steps.After the pre-amplification and subsequent library building step,whether the single cell transcriptome sequencing results can faithfully represent the original RNA expression profile before amplification remains unknow.How to reduce the amplification bias in the library building process of single-cell transcriptome is crucial for the reliability of subsequent sequencing data analysis.Over the years,there have been many methods to reduce amplification bias,mostly by physically or chemically interfering with the preferred amplification reaction process to achieve the effect of suppressing bias.However,the problem of amplification bias has always existed and has never been solved.Based on this,the limiting effect of agarose is innovatively used to improve the efficiency and uniformity of amplification reactions.Based on the principle of the limiting effect in agarose amplification methods,an efficient and low-bias transcriptional library formation scheme using agarose was optimized at the single-cell level.The main contents are divided into three parts.1.Validation of the limiting effect of agaroseAgarose,which is commonly used in molecular biology experiments,can form a porous structure in solid state and has the properties of polymer in liquid state.It has the potential to uniformly disperse the active ingredients in amplification reactions through spatial limiting effect,thus achieving the purpose of reducing bias and improving efficiency.The state changes of agarose in amplification reactions were characterized by various means.When added low melting point agarose in the reaction system of isothermal amplification or variable temperature amplification,it showed intermediate states between solidified and liquid states in the amplification reaction state.In addition,the limiting effect of agarose can be effectively verified by single molecule fluorescence resonance energy transfer experiment.2.Establishment of agarose amplification reaction systemBy adding agarose to the amplification reaction system,the concentrations of the amplified cDNA products were measured and compared with those of the amplified products without agarose.The homogeneity of amplification was compared by comparing the changes in the ratio between multiple samples after the amplification reaction.After transcriptional library building at the singlecell level of nucleic acid input,the results of library building sequencing with and without the addition of agarose in the amplification step are compared separately.In this study,agarose amplification method was proposed and established.The amplification efficiency was improved by an order of magnitude,and the amplification bias was significantly reduced.3.Application of single-cell level transcriptome sequencing technology in degenerative diseasesThe optimized amplification method was used to build a single-cell level spatial transcriptome library sequencing protocol.It was then applied to two degenerative disease models including Parkinson’s disease and retinitis pigmentosa model mice,respectively.In the application to brain diseases,the library construction scheme not only has the sampling criteria at the single-cell level,but also preserves the spatial location information of sampling which can present the expression information of Parkinson’s disease-related genes in different sampling locations in specific brain regions well.A spatial transcriptome sequencing library construction scheme was constructed of a mouse model of Parkinson’s disease based on agarose PCR,and screened out disease related high expression genes such as Mapk1,Sdha,Gnai1 and Atp6v1b2 in the striatum region.Single-cell spatial transcriptome sequencing technology has been well applied in degenerative diseases,not limited in brain diseases.It is also applicable in retinal degenerative diseases.Lowthroughput spatio-temporal transcriptome technique based on microanatomical gene expression technology is successfully constructed using improved spatial transcriptome sequencing at the singlecell level.This improved approach not only effectively preserved the expression of retinal diseaserelated genes at different locations during different stages,but also revealed the upregulation of angiogenesis-related genes at later stages of disease development.Spatial transcriptome sequencing was carried out on mice with retinal degenerative degeneration at different development stages.Different cell layer sequencing data at different positions of the retina were obtained,which showed that the angiogenesis gene Efnb2 was significantly up-regulated in the late development of retinal diseases.In general,we proposed a spatial transcriptome sequencing library building technology at the single cell scale by using the special structure and physical and chemical properties of agarose.Finally,it was applied in model mice of Parkinson’s disease and retinal degeneration.