Exonuclease III assisted target recycling amplification strategy can be used to enhance the sensitivity of mass spectrometry for the detection of short DNA sequences. However, the distribution pattern of DNA fragments produced by exonuclease III assisted target recycling amplification is generally different for samples with different concentrations of the target DNA sequence, which hinders the extraction of both qualitative and quantitative information of the target DNA from mass spectral measurements using traditional univariate or multivariate models. In this contribution, an advanced model was derived based on a reasonable assumption for the qualitative and quantitative analysis of the mass spectral measurements of DNA fragments produced by exonuclease III-assisted target recycling amplification. Experimental results demonstrated that the integration of exonuclease III assisted target recycling amplification, mass spectrometry and the advanced model could achieve sensitive and accurate quantitative results for a target short DNA sequence in complex biological medium with a detection limit of 50 pM and a mean recovery rate within the range of 89.5%–106.7%. More interestingly, the proposed model could unambiguously identify single nucleotide polymorphisms based on the distribution patterns of residual DNA fragments. Therefore, with the aid of the proposed model, mass spectrometry based on exonuclease III assisted recycling amplification has great potential for the reliable, sensitive, selective, and relatively low-cost detection and quantification of short DNA sequences in clinical diagnosis and biomedical research.
Xinxin Ren,Qingqing Zhang,Lingxia Qin,Kaiyue Hu,Zhiyong Guo,Sui Wang,Yufang Hu
In this study, a checkerboard arranged G4 nanostructure-supported electrochemical platform is developed well for the application to unique bio-enzymes examination. Herein, we focus on the two bio-enzymes involving histone acetyltransferase (HAT) and terminal deoxynucleotidyl transferase (TdT); the former leads to the acetyl transfer of acetyl coenzyme A to the lysine residue of the substrate peptide and the latter achieves the polymeric extension of DNA without template under a unique pool of dATP and dGTP (4: 6). A complex of antibody and short DNA is introduced onto the electrode surface based on the affinity interaction between acetyl in acetylated peptide and its antibody. and used for initiating reaction. Then, TdT-yielded rich-G sequence is formed to act as the backbone of checkerboard, and subsequently free G-DNAs can be stacked continually on the backbone under Mg 2+ . An excellent electrocatalytic signal to H 2 O 2 emerges noticeably, which is related with the activity of HAT p300 and TdT with a low detection limit of 2.3 pM and 0.38 mU/mL, respectively. Finally, this strategy is also used successfully for the inhibitor screening and complex sample analysis, which has important implications for the advancement of HAT- and TdT-related electrochemical bioassay and drug discovery.
危险品化学品经营许可证（带存储）