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Clinical Chemistry 0: clinchem.2004.034330v1, 2004; 10.1373/clinchem.2004.034330
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Received on March 19, 2004
Accepted on July 21, 2004

Molecular Diagnostics and Genetics

Multiplexed Genetic Analysis Using an Expanded Genetic Alphabet

Scott C. Johnson 1, David J. Marshall 1, Gerda Harms 1, Christie M. Miller 1, Christopher B. Sherrill 1, Edward L. Beaty 1, Scott A. Lederer 1, Eric B. Roesch 1, Gary Madsen 1, Gary L. Hoffman 2, Ronald H. Laessig 2, Greg J. Kopish 2, Mei Wang Baker 2, Steven A. Benner 3, Philip M. Farrell 4, James R. Prudent 1*

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.


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