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.
Subject(s)
Factor V/chemistry , Genotyping Techniques , Luciferases, Renilla/chemistry , Luminescent Proteins/chemistry , Polymorphism, Single Nucleotide , Alleles , Biomarkers/chemistry , Biotin/chemistry , Blood Coagulation Disorders/diagnosis , Calcium/metabolism , DNA/chemistry , DNA/genetics , DNA Primers/chemistry , DNA Primers/genetics , DNA-Directed DNA Polymerase/genetics , Factor V/genetics , Genome, Human , Humans , Polymerase Chain Reaction/methods , Reproducibility of Results , Sequence Analysis, DNA/methods , Streptavidin/chemistryABSTRACT
Nanodiamonds synthesized by detonation have been found not to immobilize the ring form of pUC19 plasmid DNA. Linear pUC19 molecules with blunt ends, prepared by restriction of the initial ring form of pUC19 DNA, and linear 0.25-10 kb DNA fragments are adsorbed on nanodiamonds. The amount of adsorbed linear DNA molecules depends on the size of the molecules and the size of the nanodiamond clusters.
