An advanced model for the detection of short DNA sequences by mass spectrometry based on exonuclease III assisted recycling amplification
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.
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