Initiating disease vector definition panel Figure 17 Initiating disease vector definition panel. Multiplexed Genetic Analysis Using an Expanded Genetic Alphabet 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 and isocytidine. 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: The unique genetic multiplexing platform was successfully able to test for 31 targets within the CFTR gene and provides accurate genotype assignments in a clinical setting. Few multiplexed technologies with sufficient specificity to identify small changes within the human genome are available for clinical use. Line probe or linear array technology uses nitrocellulose paper strips as the support matrix (1 ) . A benefit of the line probe is that expensive instrumentation is not required. A technology termed "oligonucleotide ligation assay" uses target-specific ligation to generate mutationspecific products that are analyzed by gel electrophoresis (2 ) . Other genetic analysis systems are coupled to microparticle flow cytometry (3) (4) (5) (6) (7) (8) . Limitations of these methods include complicated procedures that require extensive training (washings, centrifugations, and transfers), long completion times, expensive instrumentation, and technician-dependent result analysis. In addition, these technologies are not easily amenable to automation. Many hospitals and clinics currently send molecular diagnostics tests to large reference laboratories. This is in part attributable to the limitations described. Because there are benefits in having local laboratories perform diagnostic tests (9 -12 ) , there appears to be a need for molecular testing methods that circumvent the limitations posed by current testing methods.