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Detection Of Methyltransferase And Glycosylase Activity Based On Real-Time Ligase Chain Reaction

Posted on:2021-03-03Degree:MasterType:Thesis
Country:ChinaCandidate:J Y LiuFull Text:PDF
GTID:2370330620470626Subject:Analytical Chemistry
Abstract/Summary:PDF Full Text Request
DNA methyltransferase and glycosylase are two important enzymes in organisms.The abnormal expression of them may cause changes in DNA bases and even cause tumors.Therefore,developing simple and effective methods for the detection of methyltransferase and glycosylase activity has great significance for the diagnosis and treatment monitoring of diseases.Two new methods for detecting methyltransferase and glycosylase respectively was established based on real-time ligase chain reaction?real-time LCR?.The specific content is as follows:1.Detection of M.SssI methyltransferase based on real-time LCR.First,we designed two completely complementary long DNA strands,T1 and T2,which contains the5'-CCGG-3'sequence.In presence of M.SssI,the methylated long DNA strands will not be recognized and cut by HpaII and they trigger the LCR reaction as the template.The fluorescence dye,SYBR Green I,was embedded into the LCR product,enhancing the fluorescence intensity.In absence of M.SssI,the LCR reaction can not be triggered.The activity of M.SssI methyltransferase can be tested by detecting the fluorescence intensity.The linearity range is 0.002 to 0.1 U/mL and the limit of detection is 0.001 U/mL.This method can be used for screening the M.SssI inhibitors and is suitable for complex biological matrices.Our proposed method has many advantages,such as simple design,simple operation,low cost,and has great significance for clinical diagnosis and drug development.2.A method by integration of magnetic separation and real-time LCR is established to detect Uracil-DNA glycosylase?UDG?.DNA substrate containing a uracil base is conjugated on magnetic beads.At the function of UDG and endonuclease IV,a single strand DNA?ssDNA?is released from the DNA substrate and preserved in the supernatant after magnetic separation,which can be detected by LCR amplification and real-time fluorescence detection.A good linear correlation is obtained in the concentration of UDG from 5×10-4 to 5 U/mL and the limit of detection is 3x10-4 U/mL.The amplification reaction and fluorescence detection are performed simultaneously,which is easy to operate,avoids post-contamination,and widens the dynamic range.Additionally,this method enables the evaluation of inhibitors and the determination of real samples.Hence,this method provides a tool for UDG-related detection and clinical diagnosis.It is worth mentioning that this method has potential universality.On the one hand,the ssDNA template in the supernatant could be detected easily by not only LCR,but also other amplification methods.On the other hand,this method might be extended to detect various glycosylases and other DNA repair enzymes after some modification of DNA substrate,and modification of the magnetic bead with different DNA substrates can also achieve multiple detection.
Keywords/Search Tags:M.SssI methyltransferase, Uracil DNA glycosylase(UDG), Real-time ligase chain reaction(real-time LCR), SYBR Green ?
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