Development of a colorimetric test system for detection of point mutations via ligation of a tandem of short oligonucleotides on methacrylate beads

Short Oligonucleotide Tandem Ligation Assay for Genotyping of Single-Nucleotide Polymorphisms in Y Chromosome

“…In the case of the imperfect DNA template containing any base substitution in the tetranucleotide binding site, both instability of the DNA-tetranucleotide complex and the mismatch in the ligation site completely inhibit ligation of the tandem of short oligonucleotides. The fundamental difference between SOTLA and OLA approaches while using ordinary T4 DNA ligase is that the accuracy of SNPs analysis in the OLA system is ensured only by the specificity of DNA ligase while that in the SOTLA system is provided by the specificity of both ligation of threecomponent tandems and hybridization of short oligonucleotides within specific region of a DNA template [31,32].…”

“…A standard SOTLA sample consisted of polymethacrylic beads (30-60 lm, 0.5 mg) bearing immobilized 5 0 -flanking oligonucleotide (1 lmol/g) and solution (15 ll) containing tetranucleotide (10 -4 or 10 -5 M), biotinylated 3 0 -flanking oligonucleotide (10 -5 M), T4 DNA ligase (30-50 U), and DNA template (PCR product) in ligase buffer (10 mM MgCl 2 , 0.1 M NaCl, 10 mM DTT, 1 mM ATP in 20 mM Tris-HCl, pH 7.5) [32]. The preheated DNA sample (equivalent to 5-10 ll of the standard PCR mixture) was added to the reaction mixture at the last moment.…”

“…The polymer beads containing the immobilized biotinylated ligation products were washed, incubated with the Stv*AP conjugate (Sigma-Aldrich, USA) and, after additional washing, treated with BCIP/NBT chromogenic substrates (Molecular Probes, Eugene, Oregon, USA) in the manner described earlier [32].…”

“…The formation of the 20-nt ligated product was shown to occur only when the tetramer was completely complementary to its binding site on the DNA analyte. Both high selectivity of hybridization of short oligonucleotides and high specificity of ligation of three-component tandems provide the efficient discrimination of single-base substitutions in the tetramer-binding site on a DNA template [31,32].…”

Oligonucleotide probes containing polylysine residues for fabrication of DNA chips on various solid surfaces

“…Nylon membranes containing immobilized PL-oligos (10 -5 M spotting concentration) were pretreated with BSA solution (25 mg/mL) for 30 min. They were then placed into vials (1.5 mL), and ligation buffer E (50 µL) containing the DNA template (10 -7 M), tandem fragments (10 -6 M biotinylated oligonucleotide and 10 -5 M central tetranucleotide), and T4 DNA ligase (50 U) was added [29,30]. The membranes were kept at room temperature for 30 min.…”