Abstract: Calibrators for use in high-resolution melt analysis and related methods are disclosed and described. In one embodiment a polynucleotide thermal melt calibrator having a stem-loop configuration may include: a first stem region having a first nucleotide sequence; a second stem region having a second nucleotide sequence; a loop region joining the first stem region and the second stem region; and a hybridization indicator associated with at least one of the first stem region, the second stem region, or the loop region.

Abstract: Biotinidase deficiency is detected by determining the activity of the biotinidase enzyme utilizing a newborn dried blood spot and calorimetric end point analysis. The four mutations most commonly associated with complete biotinidase deficiency are G98: d7i3, Q456H, R538C, and the double mutation D444H:A171T. Partial biotinidase deficiency is almost universally attributed to the D444H mutation. To more effectively distinguish between profound and partial biotinidase deficiency, a panel of assays utilizing real time PCR and melting curve analysis is developed to detect those mutations listed above. In newborn screening for biotinidase deficiency, the analysis of common mutations is useful to distinguish between partial and complete enzyme deficiency. Combining biotinidase enzyme analysis with genotypic data also increases the sensitivity of screening for biotinidase deficiency and provides information useful to clinicians earlier than would otherwise be possible.

Abstract: A newborn screening method is provided for detecting the causes of hereditary hearing loss. Patient specimen amplicons are synthesized, wherein the amplicon is an oligonucleotide specific to a gene selected from the group consisting of cytomegalovirus (CMV), mitochondria, and connexin 26 (Cx26). They are then spotted on a substrate and immobilized as a target for microarray production as wild type and mutated alleles are allowed to hybridize thereto and undergo image analysis.

Abstract: A method is disclosed for detecting galactosemia-causing mutations in the GALT gene, comprising amplifying a portion of the GALT gene from isolated DNA and allowing a pair of labeled probes to hybridize to the portion. One of the labeled probes is adapted to match to a sequence that includes the galactosemia-causing mutation, and another of the labeled probes hybridizes to an adjacent sequence, thereby forming a hybrid. Melting curves of each hybrid are then analyzed, wherein peaks of the curves are produced at an acquired fluorescence and melting temperature, Tm; and a genotype is assigned based on the Tm of the hybrid. Resulting melting peaks are compared to reference sample peaks derived from samples characterized to contain the mutations, wherein the reference sample curves indicate a temperature change, &Dgr;Tm, between mutant and wild type peaks.

Abstract: A method and an associated microarray for detecting hemoglobinopathies by DNA microarray analysis is disclosed for a newborn screening protocol. A fragment of the human beta-globin gene is amplified and immobilized on a glass substrate and is allowed to hybridize with fluorescent dye-labeled oligonucleotide probes matched to either wild type or mutant S, C, and E alleles of the beta-globin gene. The resulting hybridized microarray slide is scanned and analyzed to reveal normal gene sequence or single nucleotide polymorphisms.