Design, Development, Validation, and Use of Synthetic Nucleic Acid Controls for Diagnostic Purposes and Application to Cystic Fibrosis Testing Todd M.

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Presentation transcript:

Design, Development, Validation, and Use of Synthetic Nucleic Acid Controls for Diagnostic Purposes and Application to Cystic Fibrosis Testing Todd M. Christensen, Mohamed Jama, Victor Ponek, Elaine Lyon, Jean Amos Wilson, Marcy L. Hoffmann, Bassem A. Bejjani The Journal of Molecular Diagnostics Volume 9, Issue 3, Pages 315-319 (July 2007) DOI: 10.2353/jmoldx.2007.060180 Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Design methodology. A: Each synthetic reference standard is designed to be amplified with a single primer pair that anneals to each primer tag region. Because the oligonucleotides are single stranded, the first round of PCR amplification generates a complementary strand for each synthetic oligonucleotide. B: After this initial cycle, the remaining PCR cycles amplify the DNA fragments in a standard manner. Reference oligonucleotides are designed for each mutation of interest. In some cases, more than one mutation may be incorporated within the same reference oligonucleotide (red circles). C: Assorted mixtures of the various reference oligonucleotides are combined and amplified using a single primer pair to produce synthetic controls carrying one or dozens of mutations that may be visualized in a single reaction tube. The Journal of Molecular Diagnostics 2007 9, 315-319DOI: (10.2353/jmoldx.2007.060180) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 PCR/OLA data. This is a comparison between the synthetic control mixture (A) and a heterozygous ΔF508 genomic control (B) with the Celera Diagnostics CFv3.0 PCR/OLA ASR reagents on an ABI 3100 genetic analyzer. The Journal of Molecular Diagnostics 2007 9, 315-319DOI: (10.2353/jmoldx.2007.060180) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 TAG-IT data. Raw data from the CF32 synthetic control and a wild-type genomic DNA sample are analyzed on the TAG-IT platform using the 40 + 4 CF ASR set. This assay does not generate a F508 wild-type signal, whereas it does produce wild-type signal for G85, G542, and 3905. These discrepancies most likely reflect the distinctiveness of the platforms themselves (ABI's OLA approach versus Tm Bioscience's allele-specific primer extension) instead of the performance of the controls. The Journal of Molecular Diagnostics 2007 9, 315-319DOI: (10.2353/jmoldx.2007.060180) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Reproducibility study. Electropherograms from 100 distinct runs of each of the CF32 control and a wild-type genomic DNA control. These runs are aligned and evaluated within a single Genotyper project file. The aligned electropherograms represent data compiled from 100 consecutive runs performed during standard testing performed at the Sacred Heart Medical Center clinical laboratory throughout the last year. The Journal of Molecular Diagnostics 2007 9, 315-319DOI: (10.2353/jmoldx.2007.060180) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions