Rapid Detection of Haptoglobin Gene Deletion in Alkaline-Denatured Blood by Loop- Mediated Isothermal Amplification Reaction Mikiko Soejima, Kouichi Egashira,

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Rapid Detection of Haptoglobin Gene Deletion in Alkaline-Denatured Blood by Loop- Mediated Isothermal Amplification Reaction Mikiko Soejima, Kouichi Egashira, Hiroyuki Kawano, Atsushi Kawaguchi, Kimitaka Sagawa, Yoshiro Koda The Journal of Molecular Diagnostics Volume 13, Issue 3, Pages 334-339 (May 2011) DOI: 10.1016/j.jmoldx.2011.01.005 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Gene structure of HP1, HP2, and HPdel, and positions of the target regions (A) and locations of two primer sets used in the LAMP reaction (B). The position and direction of each primer are shown by arrows. A downward arrow indicates the junction of gene deletion of the HPdel. The Journal of Molecular Diagnostics 2011 13, 334-339DOI: (10.1016/j.jmoldx.2011.01.005) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Results of the LAMP reaction for HPdel (left) and the 5′ region of HP exon 1 (right). A: Validation of specificity using DNA samples of known genotypes. Real-time amplification curves with turbidity detection of triplicate experiments with HP/HP, HP/HPdel, and HPdel/HPdel. The results are shown by navy (HP/HP), pink (HP/HPdel), and yellow (HPdel/HPdel). Negative control is shown by aqua. B: Validation of sensitivity using DNA samples of known genotypes. Amplification curves of triplicate experiments with various amounts of initial template DNA of HP/HPdel, 100, 10, 1, 0.1, 0.01, and 0.001 ng/reaction. The results are shown by navy (100 ng), pink (10 ng), yellow (1 ng), aqua (0.1 ng), purple (0.01 ng), and red (0.001 ng). A negative control is shown by green. C: Comparison of various preparations of blood samples. The results obtained from four different sample preparations are shown by red (1:2 dilution with water followed by heat), pink (1:10 dilution with water followed by heat), yellow (1:100 dilution with NaOH followed by heat), and aqua (1:100 dilution with NaOH). Negative control is shown by purple. The Journal of Molecular Diagnostics 2011 13, 334-339DOI: (10.1016/j.jmoldx.2011.01.005) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Evaluation of results by inspection of colors of reaction mixtures. One microliter of Fluorescent Detection Reagent (Eiken) was included in a 25-μL reaction mixture before the amplification reaction. Green and orange indicate positive and negative reactions, respectively. A 50 mmol/L NaOH-diluted blood sample of an HP/HP individual was green in the tube for HP-5′ and orange in the tube for the HP-del, that of HP/HPdel was green in both tubes, whereas that of HPdel/HPdel was green in the tube for HP-del and orange in the tube for the HP-5′. Negative controls (N.C) were orange in both tubes. The Journal of Molecular Diagnostics 2011 13, 334-339DOI: (10.1016/j.jmoldx.2011.01.005) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions