Soloviov O. Development of the test kits prototypes for DNA diagnostics of some inherited diseases (cystic fibrosis, spinal muscular atrophy, phenylketonuria)

Object - mutations in PAH, CFTR, SMN1 genes, that cause hereditary monogenic diseases: phenylketonuria, cystic fibrosis, spinal muscular atrophy.Goal is to develop the prototypes of the test kits for DNA diagnostics of the most frequent monogenic inherited disorders - cystic fibrosis, spinal muscular atrophy, phenylketonuria using the up-to-date genetic biotechnologies. Methods - isolation and purification of genomic DNA, oligonucleotide synthesis, polymerase chain reaction, restriction analysis, sequencing, different types of electrophoresis, Real-Time PCR, bioinformatic modelling, statistical analysis.Results and their novelty - methodological approaches for the detection of the most frequent mutations in CFTR (cystic fibrosis) gene using the ARMS PCR and TaqMan probes were developed. The advantages of these prototypes, compared to the existing test kits in the market, are their high efficiency and low price. Quantative DNA-analysis of the deletion in exon 7 SMN1 gene (spinal muscular atrophy) was created. Detection efficiency of the heterozygous carriers of this mutation was proved. Screening method for the detection of aberrant variants of the coding sequence of PAH gene (phenylketonuria) using danaturing gradient gel electrophoresis was elaborated. High efficiency of new dye Bava NA for the visualization of the PAAG was shown. Bank of the reference DNA samples with the mutations in PAH, CFTR, SMN1 genes, which are the inalienable components of the test kits for the DNA diagnostics of phenylketonuria, cystic fibrosis, spinal muscular atrophy, was created. The field is biotechnology.