Simple, Efficient, and Cost-Effective Multiplex Genotyping with Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of Hemoglobin.

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Presentation transcript:

Simple, Efficient, and Cost-Effective Multiplex Genotyping with Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of Hemoglobin Beta Gene Mutations Wanna Thongnoppakhun, Surasak Jiemsup, Suganya Yongkiettrakul, Chompunut Kanjanakorn, Chanin Limwongse, Prapon Wilairat, Anusorn Vanasant, Nanyawan Rungroj, Pa-thai Yenchitsomanus The Journal of Molecular Diagnostics Volume 11, Issue 4, Pages 334-346 (July 2009) DOI: 10.2353/jmoldx.2009.080151 Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic diagram of HBB gene showing distribution of 30 HBB mutations of interest and positions of tetraplex PCR products. The Journal of Molecular Diagnostics 2009 11, 334-346DOI: (10.2353/jmoldx.2009.080151) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Mass spectra of HBB mutations detected by Panel 1 and Panel 2 multiplex SBE. Representative mass spectra obtained from 21 heterozygous HBB mutation sites detected by Panel 1 multiplex SBE (1.1–1.21) and 16 by Panel 2 (2.1–2.16) are shown. Genotype of the extension product is indicated above each peak and mass value is in parentheses. Wt = wild-type. The Journal of Molecular Diagnostics 2009 11, 334-346DOI: (10.2353/jmoldx.2009.080151) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Mass spectra of 19-plex minisequencing reaction for genotyping of 21 HBB mutation sites in Panel 1 multiplex SBE and 14-plex minisequencing reaction for 16 HBB mutations in Panel 2 multiplex SBE. Panel 1 multiplex SBE: (A) c.79G>A + c.126delC (compound heterozygous) and (B) c.124_127delTTCT heterozygous. Panel 2 multiplex SBE: (C) c.124_127delTTCT heterozygous and (D) c.92 + 1G>T heterozygous. Genotype of extension product is indicated above each peak and mass value is in parentheses. Peaks labeled “Primer” indicate residual extension primers. Magnified views of heterozygote peaks are indicated by arrows and shown in insets. The Journal of Molecular Diagnostics 2009 11, 334-346DOI: (10.2353/jmoldx.2009.080151) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Mass spectra of 3-plex primer-extension reaction for genotyping in Panel 3 multiplex VSET: (A) normal, (B) c.20A>T heterozygote, (C) c.52A>T heterozygote, and (D) c.441_442insAC heterozygote. Genotype of extension product is indicated above each peak and mass value is in parentheses. Peaks labeled “Primer” indicate residual extension primers. The Journal of Molecular Diagnostics 2009 11, 334-346DOI: (10.2353/jmoldx.2009.080151) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Mismatched bases generated by extension primers used in multiplex SBE reaction and by homozygous alleles of HBB mutations. Mismatched positions near the 3′ end are likely to cause allelic loss in multiplex SBE reaction, whereas those at sixth or seventh position are not. Mismatched bases are indicated in bold and italics in box. The Journal of Molecular Diagnostics 2009 11, 334-346DOI: (10.2353/jmoldx.2009.080151) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions