The Detection Of Human Alkyladenine DNA Glycosylase Based On The Isothermal Amplification Of Nucleic Acids

Human alkyladenine DNA glycosylase?hAAG?is an important protease which can specifcally recognize and excise deoxyinosine and a variety of alkylated purines from DNA.hAAG plays significant roles in maintaining genomic integrity,and it is involved in cell cycle regulation and cell apoptosis.It is closely related to various diseases such as lung cancer and cervical cancer.In addition,hAAG is able to induce frameshift mutagenesis and microsatellite instability in human cells.Therefore,the accurate and sensitive detection of hAAG activity is crucial to both biomedical research and clinical diagnosis.In order to improve the sensitivity of detection,a variety of isothermal nucleic acid amplification techniques have been developed since the early 1990s.And they can be used for the determination of a wide range of targets,including proteins,cells,small molecules and ions.Compared with polymerase chain reaction?PCR?,isothermal nucleic acid amplification is fast and efficient,and it can be performed under simple conditions?such as water bath?achieving 10ⁿ times signal amplification at a constant temperature.Therefore,it is of great significance for low abundance detection of disease markers by isothermal nucleic acid amplification.Based on the previous works,the following major researches were carried out to detect hAAG on the basis of isothermal nucleic acid amplification.This dissertation consists of five chapters.Chapter 1:The DNA glycosylase and isothermal nucleic acid amplification are briefly introduced.The detection of DNA glycosylase based on isothermal nucleic acid amplification are highlighted.Chapter 2:A simple and sensitive fluorescence method using deoxynucleotidyl transferase?TdT?-activated endonuclease IV?Endo IV?-assisted hyperbranched signal amplification was developed for detection of hAAG.The method is extremely simple,only two nucleic acid sequences need to be designed in the reaction,and the reaction time is only¹00 min.The hairpin probe and signal probe by modifying NH₂ at 3'terminus are used to inhibit the non-specific amplification of TdT activation,and a low background signal is produced.Combined with high-efficiency hyperbranched amplification,the method has high sensitivity with detection limit of 0.09032 U/mL and shows a large dynamic range of 3 orders of magnitude from 0.1 to 50 U/mL.It can be used for accurate detection of hAAG in complex HeLa cell nuclear extract.This method can be further used to identify hAAG with other DNA glycosylases.Chapter 3:A highly sensitive method for detection DNA glycosylase based on a base excision repair mediated cascade triplet-signal amplification has been developed.The hAAG-based base excision repair mediated strand displacement amplification reaction?SDA?triggers primer-generating rolling circle amplification?PG-RCA?and Endo IV-assisted recycling signal amplification,resulting in a significantly enhanced fluorescent signal.Taking advantage of the high amplification efficiency of triple-signal amplification and the low background signal resulting from single uracil repair-mediated inhibition of nonspecific amplification,the method has high sensitivity with detection limit of 0.02612 U/mL and shows a large dynamic range of4 orders of magnitude.It can further quantify the activity of hAAG in HeLa cell extracts.Chapter 4:A label-free method based on hAAG-initiated and TdT-mediated formation of fluorescent copper nanoparticles?CuNPs?was developed to detect the activity of hAAG.TdT will initiate the template-free DNA extension by adding dTTPs along the exposed 3?-OH terminus to produce a quite long poly?T?tail,which will hybridize with the ddC-modifed assistant template and assistant probe forming stable dsDNAs with AP sites.TdT-mediated extension and Endo IV-mediated cyclic cleavage produce a large number of poly?T?tails,which will perfectly be as templates the production of fluorescent CuNPs for detection hAAG.Compared to previous fluorophore and quencher labeling methods,this method is low cost with good stability and excellent selectivity.Chapter 5:Conclusion.