Isothermal Strand-Displacement Polymerase Reaction for Visual Detection of the Southeast Asian–Type Deletion of α-Thalassemia Luxin Yu, Wei Wu, Puchang.

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Isothermal Strand-Displacement Polymerase Reaction for Visual Detection of the Southeast Asian–Type Deletion of α-Thalassemia Luxin Yu, Wei Wu, Puchang Lie, Yunhua Liu, Lingwen Zeng The Journal of Molecular Diagnostics Volume 15, Issue 6, Pages 776-782 (November 2013) DOI: 10.1016/j.jmoldx.2013.06.003 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Illustration of the production of sequence-tagged duplex DNA based on isothermal strand displacement polymerase reaction (ISDPR) and the structure of a lateral flow biosensor including the four key steps. 1: The biotin-modified hairpin recognizes and hybridizes with SEA mutant DNA when present, causing the hairpin probe to undergo stem and loop separation. 2: The primer 1 then anneals with the open stem. 3: The primer 1 triggers a polymerization reaction in the presence of dNTPs and polymerase Klenow exo−. 4: To start the next cycle, the displaced target mutant DNA strand hybridizes with another hairpin probe. NC, nitrocellulose. The Journal of Molecular Diagnostics 2013 15, 776-782DOI: (10.1016/j.jmoldx.2013.06.003) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 A: The optical responses of various concentrations of target DNA on the LFNAB with (blue bars) and without (red bars) ISDPR amplification. B: Typical photographic images and the corresponding intensities of a LFNAB in the presence of various concentrations of synthetic target DNA and the negative control. C: Photographic images and the corresponding intensities of the LFNAB tested with target DNA and target DNA including one or more mismatched bases. D: Calibration curve of a LFNAB with various concentrations of target DNA. The error bars represent SDs, n = 3. C, control line; T, test line. The Journal of Molecular Diagnostics 2013 15, 776-782DOI: (10.1016/j.jmoldx.2013.06.003) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 The sensitivity of the prepared lateral flow biosensor (LFB) for human genomic DNA. A: Typical photographic images and the corresponding intensities visualized on a LFNAB in the presence of various concentrations of genomic DNA from a SEA patient and a negative control. B: Calibration curve of the LFB prepared using various concentrations of genomic DNA from a SEA patient. The error bars represent the SDs, n = 3. C, control line; T, test line. The Journal of Molecular Diagnostics 2013 15, 776-782DOI: (10.1016/j.jmoldx.2013.06.003) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 A: Typical photographic images and the corresponding intensities obtained using a lateral flow biosensor (LFB) for the detection of Southeast Asian (SEA) patient genomic DNA; α3.7, α4.2, and β-thalassemia patients’ genomic DNA and the genomic DNA of a healthy person were used as negative controls. B: Gel electrophoresis of amplified PCR products obtained by gap-PCR. Lane M contains a DL2000 molecular weight marker; lanes 1,4, and 8: genomic DNA from three SEA patients; lanes 2 and 3: genomic DNA from healthy humans; and lanes 5, 6, and 7: genomic DNA from patients with α3.7, α4.2, and β-thalassemia, respectively. C, control line; T, test line. The Journal of Molecular Diagnostics 2013 15, 776-782DOI: (10.1016/j.jmoldx.2013.06.003) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions