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| Detection of MicroRNA Using an Exponential Nucleic Acid Isothermal Amplification Strategy Based on a DNA “Y” Structure |
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| DOI： |
| KeyWord:miRNA isothermal exponential amplification reaction(EXPAR) sequence dependent disease diagnosis |
| Author | Institution |
| ZHENG Xu-ling,YIN Wen,CHEN Jun,LI Yu,DAI Zong,ZOU Xiao-yong |
1.School of Chemistry,Sun Yat-sen University,Guangzhou,China;2.School of Biomedical Engineering,Sun Yat-sen University,Guangzhou,China;3.School of Pharmaceutical Sciences,Southern Medical University,Guangzhou,China |
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| Abstract: |
| A rapid and low sequence-dependent miRNA detection method was established based on a “Y” DNA structure and a signal amplification function of isothermal exponential amplification reaction(EXPAR),which was named as Y-EXPAR.This method is easy to operate,with no need for DNA template labeling and a temperature change process.Meanwhile,the miRNA analysis was combined with fluorescence analysis technology,with Let-7b as a model target for miRNA detection because of its important role in cellular processes and human disease.According to the sequence of the model miRNA,two partially complementary DNA templates were designed to form a “Y” shaped structure together with the target miRNA,which could mediate a bidirectional amplification resulting in high efficiency and a short reaction time of 10 min.Furthermore,the results generated a good anti-interference for homologous sequences,distinguishing the let-7b from the let-7 homologous sequences with differences of 1-4 bases.Next,when used to detect other miRNAs with a lot of sequence changes(GC content),this “Y” template could also keep a good amplification performance,achieving the reduce of the dependence of amplification performance on template sequence.To achieve the best amplification efficiency,the reaction temperature,the dosage of endonuclease and polymerase were optimized.Under the optimal experimental conditions that the reaction temperature was 45 ℃,the concentration of Nt.BstNBI endonuclase was 0.04 U/μL,and the concentration of Vent(exo-) DNA polymerase was 0.06 U/μL,this method obtained a limit of detection down to 0.3 amol/L(corresponding to 1.8 copies of let-7b per 10 μL).And the linearity was maintained well within the range of 10 concentration orders.Results of the application in let-7b detection of tumor cell lysate showed a distinct difference between the POI values of 1-1 000 cells and the blank sample.And there was a linear relationship between the POI value and the logarithmic value of the number of cells.These results indicate that Y-EXPAR has an advantage in the detection of miRNA in actual samples.Benefited from the high efficiency,specificity,anti-interference and low sequence dependence of the amplification,this method provides a new idea for the rapid nucleic acid constant temperature amplification,and has a good application prospect in the research and clinical diagnosis of miRNA-related diseases. |
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