ABSTRACT
In this study, essential test characteristics of the recently described multiplex ligation-dependent probe amplification (MLPA) method are presented, using chromosome 22 as a model. This novel method allows the relative quantification of approximately 40-45 different target DNA sequences in a single reaction. For the purpose of this study, MLPA was performed in a blinded manner on a training set containing over 50 samples, including typical 22q11.2 deletions, various atypical deletions, duplications (trisomy and tetrasomy), and unbalanced translocations. All samples in the training set have been previously characterized by fluorescence in situ hybridization (FISH) with cosmid or BAC clones and/or cytogenetic studies. MLPA findings were consistent with cytogenetic and FISH studies, no rearrangement went undetected and repeated tests gave consistent results. At a relative change in comparative signal strength of 30% or more, sensitivity and specificity values were 0.95 and 0.99, respectively. Given that MLPA is likely to be used as an initial screening method, a higher sensitivity, at the cost of a lower specificity, was deemed more appropriate. A receiver operator characteristic (ROC) curve analysis was performed to calculate the most optimal threshold range, with associated sensitivity and specificity values of 0.99 and 0.97, respectively. Finally, performance of each individual probe was analyzed, providing further useful information for the interpretation of MLPA results. In conclusion, MLPA has proven to be a highly sensitive and accurate tool for detecting copy number changes in the 22q11.2 region, making it a fast and economic alternative to currently used methods. The current study provides valuable and detailed information on the characteristics of this novel method.
Subject(s)
Chromosome Aberrations , Chromosomes, Human, Pair 22 , Cytogenetic Analysis/methods , DiGeorge Syndrome/diagnosis , Polymerase Chain Reaction/methods , Cell Line , Gene Dosage , Humans , Reproducibility of ResultsABSTRACT
Traditional approaches to phylogeny reconstruction have not allowed precise resolution of the evolutionary relationships between the major deuterostome phyla (chordates, hemichordates, echinoderms). Here we report the use of a molecular approach to investigate deuterostome phylogeny. We have used a polymerase chain reaction-based strategy to amplify, clone and sequence parts of the genes coding for 18S ribosomal RNA from Saccoglossus cambrensis (Hemichordata), Arbacia sp. (Echinodermata) and, for comparison, Mytilus edulis (Mollusca). We report the results of phylogenetic reconstructions using these, and homologous sequences from other eukaryotes. The results of our analyses are consistent with the hypothesis that S. cambrensis and vertebrates share a common ancestor not shared by echinoderms.
