Short Oligonucleotide Tandem Ligation Assay for Genotyping of Single-Nucleotide Polymorphisms in Y Chromosome |
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Authors: | Larisa M Skobeltsyna Dmitrii V Pyshnyi Eugenia M Ivanova Vadim A Stepanov Valery P Puzyrev Grigory M Dymshits Vladimir N Kharkov Valentina F Zarytova |
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Institution: | (1) Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentieva ave., 8, Russian Federation, 630090 Novosibirsk, Russia;(2) Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia;(3) Institute of Medical Genetics, Siberian Branch of the Russian Academy of Medical Sciences, Tomsk, Russia; |
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Abstract: | We propose a novel universal methodology, Short Oligonucleotide Tandem Ligation Assay (SOTLA), for SNP genotyping. SOTLA is
based on using a tandem of short oligonucleotide (TSO) probes consisting of three fragments: the core oligonucleotide and
two flanking oligomers, one of which is immobilized onto a solid support and another one contains the biotin label. TSO is
self-associated on a complementary DNA template, forms the complex containing two nicks, which are efficiently ligated with
DNA ligase giving biotinylated oligonucleotide covalently bound to polymer beads. No ligation of TSO on an imperfect DNA template
bearing the base substitution in the core binding site is occurred. We used SOTLA for the highly selective SNP analysis in
different DNA fragments of human Y chromosome. Comparison of SOTLA results with those of PCR-RFLP and allele-specific PCR
techniques demonstrates that SOTLA ensures the univocal reliable SNP analysis in different PCR fragments varying in length
and base composition. The fundamental difference between SOTLA and well known OLA approaches while using T4 DNA ligase is
that the accuracy of SNP analysis in OLA is ensured only by the specificity of ligase while that in SOTLA is provided by the
specificity of both ligation and hybridization of TSO probes. |
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