The development of the ACADVL scanning assay using a two-step PCR and high resolution melting analysis
MetadataShow full item record
Type of Workapplication/pdf
vii, 65 pages
ProgramTowson University. Forensic Science Program
RightsCopyright protected, all rights reserved.
There are no restrictions on access to this document. An internet release form signed by the author to display this document online is on file with Towson University Special Collections and Archives.
Very long chain acyl-CoA dehydrogenase deficiency is the second most common fatty acid oxidation disorder detected by newborn screening. Currently, tandem mass spectrometry is used as the primary detection method, followed by molecular genetic testing to confirm the presence of a mutation. A simpler, more cost effective method of diagnosis is high resolution melting (HRM) following amplification of the target. HRM does not require physical processing or separation steps. In this study, PCR primers' that amplify each of the 20 exons that make up the ACADVL gene were designed. One primer pair for each exon was selected based on PCR performance and target specificity. After defining the PCR cycling conditions, we performed a blinded study with 30 different genomic DNA samples. Synthetic constructs were used as positive controls for each test. Seven different mutations were detected from the high resolution melting profiles. Bi-directional DNA sequencing (Sanger Sequencing) confirmed the mutations.