Studies On Analysis Of Single Nucleotide Polymorphism,mRNA Splicing Variants,and Telomerase Activity Based On Nucleic Acid Amplification

Gene expression,that is,genetic information encoded in DNA is transcribed to messenger RNA(mRNA)and then mRNA is translated to protein,is the central dogma of molecular biology.With the completion of Human Genome Project(HGP)and rapid developing of next generation sequencing(NGS)technologies,the determination of biomarkers such as DNA variation,epigenetic elements,gene expression,and activity of some proteins have become a hot research field.Changes of these biomarkers are closely associated with the occurrence of several diseases,especially cancers.Thus,the simple,rapid,sensitive,and specific analysis methods have become increasingly important because of their great significance for studies of the basic process of life,genetic disease,medical diagnostics as well as target-drugs discovery.In this dissertation,we developed simple,rapid,sensitive,and specific methods for detecting single nucleotide polymorphism(SNP),alternative splicing variants,and telomerase activity by nucleic acid amplification.SNP refers to the diversity of DNA sequences,which caused by a single base change in some specific loci of genome.Assay of sequence-specific nucleic acids and SNP are very important for gene function research and hereditary disease diagnosis.Developing rapid,simple,specific and highly sensitive methods for determination of sequence-specific nucleic acids and SNP in homogeneous solution with label-free fashion have wide application prospects.In this research,we designed two stem-loop probes,which can hybridize with target sequence.In the presence of thermostable DNA ligase,stem-loop probes can be ligated by using target sequence as a template and form double stem-loop structure.Once the double stem loop structure formed,the auto-cycling strand displacement DNA synthesis can be repeated continuously and leads to quickly exponential amplification under isothermal conditions in the presence of primers,dNTPs,and Bst DNA polymerase with high-displacement activity.The LAMP reaction could produce a large amount of DNA,which can form large particles in acidic conditions.The large particles result in very strong light scattering which intensity is well proportional to the concentration of DNA.With the proposed method,10 aM mutant DNA can be detected and as low as 0.1%mutation DNA can be discriminated in the presence of a large excess of wild-type DNA.Alternative splicing is a central regulated process during gene expression that greatly increased the structural and functional biodiversity of proteins coding by different mRNA isoforms.Alternative splicing variants are crucial in many cellular processes.Aberrant alternative splicing and changes in the expression levels are implicated in a variety of disease and the development of cancers.Given the vital role of alternative splicing in regulating cellular function,detection and quantification of alternative splicing variants could facilitate to understanding the regulatory function of alternative splicing variants in disease,cellular protein diversity,genetic diagnosis and gene therapies.Breast cancer susceptibility gene 1(BRCA1)encodes a nuclear phosphoprotein that plays an important role in maintaining genomic stability,and it also acts as a tumor suppressor.Alternative splicing of BRC A1 is used as an indicator of malignant breast cancer.In this research,we designed specific DNA probes near the junction sites of different alternative splicing variants of BRCA1 mRNA,the DNA probes can be ligated by using mRNA as the template.With PCR amplification of the the ligated DNA probe,different alternative splicing variants of BRCA1 mRNA can be sensitively detected with high specificity.Moreover,by ligation-based PCR,multiple alternative splicing variants can be simultaneously determined in one reaction.Telomerase is a ribonucleoprotein with reverse transcriptase activity,which can add the repeats(TTAGGG)to the ends of chromosomes.Telomerase is repressed in most normal somatic cells but is reactivated in the majority of tumor cells,which allows telomerase length maintenance and cell immortalization and has been considered involvement in carcinogenesis.It is a crucial biomarker for cancers.Its reliable and sensitive detection,especially in a single cell,has great significance for early diagnosis of cancers and studies of tumor progression and anticancer therapy,but still remains challenging due to the nonspecific amplification.Here we have developed a novel stem-loop primer-mediated exponential amplification(SPEA),which can highly specifically and efficiently amplify the telomerase-elongated telomerase repeat with near zero nonspecific signal.The SPEA-based assay thus can accurately detect telomerase activity in crude lysate of a single cell and suited to detect the cellular heterogeneity arising from cell-to-cell variations.