1. The Suitability of Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry in a Laboratory Developed Test Using Cystic Fibrosis Carrier Screening as a Model 
Daniel H. Farkas, Nicholas E. Miltgen, Jay Stoerker, Dirk van den Boom, W. Edward Highsmith, Lesley Cagasan, Ron McCullough, Reinhold Mueller, Lin Tang, John Tynan, Courtney Tate, Allan Bombard 
The Journal of Molecular Diagnostics 
Volume 12, Issue 5, Pages (September 2010)
DOI: /jmoldx
Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2. Figure 1
Mass spectra of ΔF508 (DF508) assay representative of SBE/iPlex chemistry. A: “No amplification” control (herring sperm DNA) shows no base extension of the extension primer (mass ≈ 6079). B: Validation sample (GGC-068) genotyped as normal for the ΔF508 assay; only the base, “A,” corresponding to the normal allele, was added resulting in the peak labeled wild-type (WT; mass ≈ 6375); note the absence of any extension product at the mass labeled with the mutant (MUT) dotted line. C: Validation sample (GGC-072) genotyped as heterozygous for ΔF508 as both the WT base, “A,” and mutant base, “T,” were added to the extension primer. D: Validation sample (WU-026) genotyped as homozygous mutant for ΔF508 as only the mutant base “T” was added to the extension primer. Peaks in the spectra above (asterisks) are the extension primer of another assay in the “plex” that coincidentally is in the same mass “window.” X axis, m/z in Da; y axis, relative intensity (software calculates values from raw data). UEP, unextended primer.
The Journal of Molecular Diagnostics  , DOI: ( /jmoldx )
Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3. Figure 2
Mass spectra of 3905insT assay representative of hME chemistry. A: Validation sample (GGC-033) genotyped as normal for 3905insT; only the wild-type (WT) bases “G and C” were added. B: Validation sample (WU-085) genotyped as heterozygous for 3905insT; WT bases “G” and “C” and mutant bases “A, G, and C” were added to the extension primer. Peak in the spectra (asterisk) in A is the extension primer of another assay in the “plex” that coincidentally is in the same mass “window.” X axis, mass, in Da; y axis, intensity in arbitrary units.
The Journal of Molecular Diagnostics  , DOI: ( /jmoldx )
Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4. Figure 3
The three assays (CFTRdele2_3, CFTR_WT_2, and CFTR_WT_3) are used to identify the deletion of exons two and three of the CFTR gene and to determine the zygosity of this deletion. A mass spectrum of a patient (WU-045) without the CFTR deletion on either allele is shown in A. The PCR primers for the CFTRdele2_3 assay are designed such that amplification will not occur in the presence of the two exons (A). In the absence of the deletion, the primers used for its amplification cannot anneal because their complementary target is absent.14 Therefore, in a truly normal patient, no base extension can occur. An example of a specimen (NCH-045) with the deletion of exons two and three on at least one allele is shown in B. To determine the zygosity of this specimen, the mass spectra for assays CFTR_WT_E2 and CFTR_WT_E3 must be analyzed to confirm the presence of these exons on the other allele. This specimen (NCH-045) clearly showed base extension of these other assays C and D, making the genotype heterozygous for the deletion detected in B. Had there been no base extension for NCH-045 in spectra C and D, this specimen would be homozygous for the deletion observed in B. Peaks in the spectra in A and B (asterisks) is the extension primer of another assay in the “multiplex” that coincidentally is in the same mass “window.”
The Journal of Molecular Diagnostics  , DOI: ( /jmoldx )
Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions