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

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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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Abbreviations

SOTLA:

Short oligonucleotide tandem ligation assay

Bio:

Biotin

BCIP:

5-bromo-4-chloro-3-indolylphosphate

NBT:

Nitro blue tetrazolium

Stv–AP:

Streptavidin–alkaline phosphatase conjugate

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Acknowledgments

We thank T. Bushueva for the great technical assistance. This work was supported in part by grants from the Russian Foundation for Basic Research (06-04-49263-a, 05-04-08040-ofi-a, 08-04-12041-ofi, 06-04-48274-a), from MESI of RF (02.512.11.2226), from the President of Russian Federation (MD-88.2003.04), from Wenner-Gren foundation for Anthropological Research (6801), from MCB program of RAS, and by cross-disciplinary grants from SB RAS (61,76).

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Authors and Affiliations

  1. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentieva ave., 8, Russian Federation, 630090, Novosibirsk, Russia

    Larisa M. Skobeltsyna, Dmitrii V. Pyshnyi, Eugenia M. Ivanova & Valentina F. Zarytova

  2. Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia

    Larisa M. Skobeltsyna & Grigory M. Dymshits

  3. Institute of Medical Genetics, Siberian Branch of the Russian Academy of Medical Sciences, Tomsk, Russia

    Vadim A. Stepanov, Valery P. Puzyrev & Vladimir N. Kharkov

Authors
  1. Larisa M. Skobeltsyna
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  2. Dmitrii V. Pyshnyi
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  3. Eugenia M. Ivanova
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  4. Vadim A. Stepanov
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  5. Valery P. Puzyrev
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  6. Grigory M. Dymshits
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  7. Vladimir N. Kharkov
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  8. Valentina F. Zarytova
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Correspondence to Valentina F. Zarytova.

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Oligodeoxyribonucleotides are abbreviated without prefix “d”.

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Skobeltsyna, L.M., Pyshnyi, D.V., Ivanova, E.M. et al. Short Oligonucleotide Tandem Ligation Assay for Genotyping of Single-Nucleotide Polymorphisms in Y Chromosome. Mol Biotechnol 45, 1–8 (2010). https://doi.org/10.1007/s12033-009-9208-2

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