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Received on March 19, 2004
Accepted on July 21, 2004
Molecular Diagnostics and Genetics |
1 Eragen Biosciences, Inc., Madison, WI
2 Wisconsin State Laboratory of Hygiene, Madison, WI
3 Department of Chemistry, University of Florida, Gainesville, FL
4 Department of Pediatrics, University of Wisconsin, Madison, WI
* To whom correspondence should be addressed. E-mail: jprudent{at}eragen.com.
Background: All states require some kind of testing for newborns, but the policies are far from standardized. In some states, newborn screening may include genetic tests for a wide range of targets, but the costs and complexities of the newer genetic tests inhibit expansion of newborn screening. We describe the development and technical evaluation of a multiplex platform that may foster increased newborn genetic screening.
Methods: MultiCode® PLx involves three major steps: PCR, target-specific extension, and liquid chip decoding. Each step is performed in the same reaction vessel, and the test is completed in 
3 h. For site-specific labeling and room-temperature decoding, we use an additional base pair constructed from isoguanosine (iG) and isocytidine (iC). We used the method to test for mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The developed test was performed manually and by automated liquid handling. Initially, 225 samples with a range of genotypes were tested retrospectively with the method. A prospective study used samples from >400 newborns.
Results: In the retrospective study, 99.1% of samples were correctly genotyped with no incorrect calls made. In the perspective study, 95% of the samples were correctly genotyped for all targets, and there were no incorrect calls.
Conclusions: Using one additional base pair not found in nature, we developed a genetic multiplexing platform and used it to test for 31 targets within the CFTR gene with high precision in a clinical setting.
The following articles in journals at HighWire Press have cited this article:
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  M. B. Miller and Y.-W. Tang Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology Clin. Microbiol. Rev., October 1, 2009; 22(4): 611 - 633. [Abstract] [Full Text] [PDF]
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F. S. Nolte, D. J. Marshall, C. Rasberry, S. Schievelbein, G. G. Banks, G. A. Storch, M. Q. Arens, R. S. Buller, and J. R. Prudent MultiCode-PLx System for Multiplexed Detection of Seventeen Respiratory Viruses J. Clin. Microbiol., September 1, 2007; 45(9): 2779 - 2786. [Abstract] [Full Text] [PDF]
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W.-M. Lee, K. Grindle, T. Pappas, D. J. Marshall, M. J. Moser, E. L. Beaty, P. A. Shult, J. R. Prudent, and J. E. Gern High-Throughput, Sensitive, and Accurate Multiplex PCR-Microsphere Flow Cytometry System for Large-Scale Comprehensive Detection of Respiratory Viruses J. Clin. Microbiol., August 1, 2007; 45(8): 2626 - 2634. [Abstract] [Full Text] [PDF]
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Z. Yang, A. M. Sismour, P. Sheng, N. L. Puskar, and S. A. Benner Enzymatic incorporation of a third nucleobase pair Nucleic Acids Res., July 26, 2007; 35(13): 4238 - 4249. [Abstract] [Full Text] [PDF]
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Z. Yang, D. Hutter, P. Sheng, A. M. Sismour, and S. A. Benner Artificially expanded genetic information system: a new base pair with an alternative hydrogen bonding pattern Nucleic Acids Res., December 4, 2006; 34(21): 6095 - 6101. [Abstract] [Full Text] [PDF]
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 M. J. Moser, D. R. Christensen, D. Norwood, and J. R. Prudent Multiplexed Detection of Anthrax-Related Toxin Genes J. Mol. Diagn., February 1, 2006; 8(1): 89 - 96. [Abstract] [Full Text] [PDF]
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L. A. Ugozzoli Multiplex Assays with Fluorescent Microbead Readout: A Powerful Tool for Mutation Detection Clin. Chem., November 1, 2004; 50(11): 1963 - 1965. [Full Text] [PDF]
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