title: [Bioluminescent reporters to identify gene allelic variants].
abstract: The method of single nucleotide polymorphism identification based on primer extension reaction (PEXT) with the following bioluminescent solid-phase microassay was developed. The recombinant Ca2+-regulated photoprotein obelin and coelenterazine-dependent luciferase Renilla muelleri were used as reporters. Factor V Leiden polymorphism 1691 G-->A (R506Q) of human F5 gene genotyping was used for investigation. Genomic DNA was amplified by PCR using primers, flanking polymorphic site of 140 base pairs. PCR products were used as a template for two PEXT reaction using two primers with 3'-end nucleotides, complementary either normal or mutant alleles. At complementarity of template and allelic-typical primer its extension with DNA-polymerase takes place. The products carried biotin due to availability ofbiotinylated dUTP in the reactions mixture. The assay was carried out using obelin-streptavidin chemical conjugates. Optimal PEXT-reaction conditions providing high reliability of SNP genotyping were found. A new approach to determine both alleles in one well was developed applying two bioluminescent reporters. Availability of the proposed approach was shown in the study of clinical DNA samples.
authors.forename: V V
authors.initials: VV
authors.lastname: Krasitskaia
authors.forename: L P
authors.initials: LP
authors.lastname: Burakova
authors.forename: I A
authors.initials: IA
authors.lastname: Pyshnaia
authors.forename: L A
authors.initials: LA
authors.lastname: Frank
mesh_terms: D000483:Alleles; D015415:Biomarkers / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D001778:Blood Coagulation Disorders / Q000175:diagnosis; D002118:Calcium / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D005165:Factor V / Q000737:chemistry* / Q000235:genetics; D015894:Genome, Human; D060005:Genotyping Techniques; D006801:Humans; D049410:Luciferases, Renilla / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods; D019809:Streptavidin / Q000737:chemistry
chemical_list: D015415:Biomarkers; D017931:DNA Primers; D008164:Luminescent Proteins; C095381:factor V Leiden; C010497:obelin; D001710:Biotin; D005165:Factor V; D004247:DNA; D019809:Streptavidin; D049410:Luciferases, Renilla; D004259:DNA-Directed DNA Polymerase; D002118:Calcium