Multiplexed genetic analysis using an expanded genetic alphabet

Abstract

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 approximately 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.

Figure 1.

Molecular structure of diGTP-biotin used in the described method.

Figure 2.

Visual interface using the MultiCode analysis software developed for the CFTR mutation analysis test. The data are displayed in various forms: a scatter plot with raw MFI (top left); MFI fractions (top middle); an individual sample summary, which can include mutation determination and specific numerical MFI (top right); and graphical determinations for all samples and targets analyzed (bottom). WT, wild type.

Figure 3.

Schematic of MultiCode PLx platform format. (Step 1), target regions are amplified by PCR to produce amplicons with iC attached to their 5′ ends. (Step 2), tagged target-specific extenders are site-specifically labeled with diGTP-label when the correct amplicon is present. For this study, the label was biotin, and streptavidin-phycoerythrin was used for signaling purposes. (Step 3), the tags are captured on EraCode-modified Luminex microspheres. The captured reactions are analyzed with the Luminex¹⁰⁰ system.