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Panel of X-linked single-nucleotide polymorphic markers for DNA identification (XSNPid) based on multiplex genotyping by multilocus PCR and MALDI-TOF mass spectrometry


Human genetic markers linked with the X chromosome (X-linked) are used in the field of population and medical genetics, as well as for DNA identification of individuals in forensic science and forensic medicine. We proposed an XSNPid panel that consists of 66 unlinked single nucleotide X chromosome markers and developed a protocol for their multiplex genotyping using multilocus PCR and MALDI-TOF mass spectrometry. The XSNPid panel is genotyped within two multiplexes (36 and 30 markers). The developed protocol provides an efficient genotype reading; the fraction of determined genotypes is 98.29%. The high level of gene diversity (0.461) for the X-linked SNPs included in the panel is characteristic of the Russian population. A total of 63 out of 66 markers that provide a high efficiency of genotyping and independent inheritance are suitable for DNA identification purposes. The XSNPid panel is characterized by a very high discriminating ability when studying the Russian population. The probability of genotype coincidence in two unrelated individuals is 9 × 10–27 for women and 2 × 10–18 for men. Also, the XSNPid panel has a greater multiplex capacity in addition to a higher discriminating ability compared to the other closest analogues of the X chromosome SNP sets, which makes it more cost effective and less time consuming. The XSNPid panel is a convenient tool, not only for individual DNA identification, but also for population genetic studies.

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matrix assisted laser desorption/ionisation-time of flight


single nucleotide polymorphism


  1. 1.

    Stepanov V.A. 2002). Etnogenomika naseleniya Sibiri i Srednei Azii (Ethnogenomics of the Population of Siberia and Central Asia). Tomsk: Pechatnaya Manufaktura.

    Google Scholar 

  2. 2.

    Stepanov V.A., Khar’ kov V.N., Puzyrev V.P. 2006. The evolution and phylogeography of human Y-chromosome lineages. Inf. Vestn. Vavilovskogo O-va. Genet. Sel. 10, 57–73.

    Google Scholar 

  3. 3.

    Khitrinskaya I.Yu., Khar’kov V.N., Stepanov V.A. 2010. Genetic diversity of the chromosome X in aboriginal Siberian populations: The structure of linkage disequilibrium and haplotype phylogeography of the ZFX locus. Mol. Biol. (Moscow). 44 (5), 709–719.

    CAS  Article  Google Scholar 

  4. 4.

    Vagaitseva K.V., Kharkov V.N., Cherpinskaya K.V., Khitrinskaya I.Yu., Stepanov V.A. 2015. Genetic variability of X-linked STR markers in Siberian populations. Mol. Biol. (Moscow). 49 (2), 267–274.

    CAS  Article  Google Scholar 

  5. 5.

    Dobyns W.B., Filauro A., Tomson B., Chan A.S., Ho A.W., Ting N.T., Oosterwijk J.C., Ober C. 2004. Inheritance of most X-linked traits is not dominant or recessive, just X-linked. Am. J. Med. Genet. 129, 136–143.

    Article  Google Scholar 

  6. 6.

    Burdett T., Hall P.N., Hasting E., Hindorff L.A., Junkins H.A., Klemm A.K., MacArthur J., Manolio T.A., Morales J., Parkinson H., Welter D. 2015. A Catalog of Published Genome-Wide Association Studies. wwwgenomegov/gwastudies. Accessed July 18, 2015.

    Google Scholar 

  7. 7.

    Szibor R. 2007. X-chromosomal markers: Past, present and future. Forensic Sci. Int. Genet. 1, 93–99.

    Article  PubMed  Google Scholar 

  8. 8.

    Lancia M., Severini S., Coletti A., Margiotta G., Dobosz M., Carnevali E. 2011. Using X-chromosomal markers in rape investigation. Forensic Sci. Int. Genet. 3, e55–e56

    Article  Google Scholar 

  9. 9.

    Stepanov V.A., Balanovskii O.P., Melnikov A.V., Kharkov V.N., Tyazhelova T.V., Akhmetova V.L., Zhukova O.V., Shneider Yu.V., Shil’nikova N.N., Borinskaya S.A., Marusin A.V., Spiridonova M.G., Simonova K.V., Khitrinskaya I.Yu., Radzhabov M.O., et al. 2011. Characteristics of populations of the Russian Federation over the panel of fifteen loci used for DNA identification and in forensic medical examination. Acta Naturae. 3, 59–71.

    Google Scholar 

  10. 10.

    Stepanov V.A., Melnikov A.V., Lash-Zavada A.Y., Kharkov V.N., Borinskaya S.A, Tyazhelova T.V., Zhukova O.V., Schneider Y.V., Shil’nikova I.N., Puzyrev V.P., Rybakova A.A., Yankovsky N.K. 2010. Genetic variability of 15 autosomal STR loci in Russian populations. Legal Medicine. 12, 256–258.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Shorokhova D.A., Stepanov V.A., Udovenko Yu.D., Hovoselov V.P., Puzyrev V.P. 2005. Genetic variation and discriminating power of four DNA microsatellites in the Russian population. Mol. Biol. (Moscow). 39 (6), 845–850.

    CAS  Article  Google Scholar 

  12. 12.

    Costa A., Morais P., Vieira da Silva C., Matos S., Marques dos Santos R., Espinheira R, Costa Santos J., Amorim A. 2014. X-chromosome STR markers data in a Cabo Verde immigrant population of Lisboa. Mol. Biol. Rep. 41, 2559–2569.

    CAS  Article  Google Scholar 

  13. 13.

    Gill P., Ivanov P.L., Kimpton C., Piercy R., Benson N., Tully G., Evett I., Hagelberg E., Sullivan K. 1994. Identification of the remains of the Romanov family by the DNA analysis. Nat. Genet. 6, 130–135.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Gill P., Werrett D.J., Budowle B., Guerrieri, R. 2004. An assessment of whether SNPs will replace STRs in national DNA databases: Joint considerations of the DNA working group of the European Network of Forensic Science Institutes (ENFSI) and the Scientific Working Group on DNA Analysis Methods (SWGDAM). Sci. Justice. 44, 51–53.

    Article  PubMed  Google Scholar 

  15. 15.

    Krawczak M. 1999. Informativity assessment for biallelic single nucleotide polymorphisms. Electrophoresis. 20, 1676–1681.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Li C., Zhang S., Zhao S., Liu Y., Lin Y. 2010. Analysis of 14 highly informative SNP markers on X chromosome by TaqMan SNP genotypoing assay. Forensic Sci. Int.: Genetics. 4, e145–e148.

    Article  Google Scholar 

  17. 17.

    Tomas C., Sanchez J.J., Castro J.A., Børsting C., Morling N. 2010. Forensic usefulness of a 25 X-chromosome single-nuleotide polymorphism set. Transfusion. 50, 2258–2265.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Pereira V., Tomas C., Amorim A., Morling N., Gusmao L., Prata M.J. 2011. Study of 25 X-chromosome SNPs in Portugese. Forensic Sci. Int.: Genetics. 5, 336–338.

    CAS  Article  Google Scholar 

  19. 19.

    Berglund E.C., Kiialainen A., Syvänen A.-C. 2011. Next-generation sequencing technologies and applications for human genetic history and forensics. Invest. Genet. 2, 23.

    CAS  Article  Google Scholar 

  20. 20.

    Yaran Yang, Bingbing Xie, Jiangwei Yan. 2014. Application of next-generation sequencing technology in forensic science. Genom. Proteom. Bioinform. 12, 190–197.

    Article  Google Scholar 

  21. 21.

    Churchill J.D., Chang J., Ge J., Rajagopalan S.C. Wootton C.W. 2015. Blind study evaluation illustrates utility of the Ion PGM system for use in human identity DNA typing. Croat. Med. J. 56, 218–229.

    Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Stepanov V.A., Trifonova E.A. 2013. Multiplex SNP genotyping by MALDI–TOF mass spectrometry: Frequencies of 56 immune response gene SNPs in human populations. Mol. Biol. (Moscow). 47, 852–862.

    CAS  Article  Google Scholar 

  23. 23.

    Electrospray and MALDI Mass Spectrometry: Fundamentals, Instrumentation, Practicalities, and Biological Applications, 2nd ed. Ed. Cole R.B. Wiley, 2012.

  24. 24.


  25. 25.


  26. 26.

    Guo S., Thompson E. 1992. Performing the exact test of Hardy–Weinberg proportion for multiple alleles. Biometrics. 48, 361–372.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Trifonova E.A., Eremina E.R., Urnov F.D., Stepanov V.A. 2012. Genetic diversity and structure of the MTHFR gene linkage disequilibrium in populations of northern Eurasia. Acta Naturae. 4, 80–96.

    Google Scholar 

  28. 28.

    Stepanov V.A., Kharkov V.N., Trifonova E.A., Marusin A.V. 2014). Metody statisticheskogo analiza v populyatsionnoi i evolyutsionnoi genetike cheloveka. Uchebno-metodicheskoe posobie (Methods of Statistical Analysis in Human Population and Evolutionary Genetics: A Manual), Ser. Nasledstvennost’ i zdorov’e (Heredity and Health Book Series), vol. 12, Tomsk: Pechatnaya Manufaktura.

    Google Scholar 

  29. 29.

    Slatkin M. 1994. Linkage disequilibrium in growing and stable populations. Genetics 137, 331–336.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Excoffier L., Lischer H. 2011). Arlequin v. 3.5: An Integrated Software Package for Population Genetics Data Analysis. Swiss Institute of Bioinformatics.

  31. 31.

    Validation Guidelines for DNA Analysis Methods, Scientific Working Group on DNA Analysis Methods, 2012.

  32. 32.

    Promega. 2013). Internal Validation Guide of Autosomal STR Systems for Forensic Laboratories: Reference Manual.

  33. 33.

    https://worldwidepromegacom/products/geneticidentity/ str-analysis-for-forensic-and-paternity-testing/ powerplex-fusion-str-kits/

  34. 34.

    Sanchez J.J., Phillips C., Borsting C., Balogh K., Bogus M., Fondevila M., Harrison C.D., Musgrave-Brown E., Salas A., Syndercombe-Court D., Schneider P.M., Carracedo A., Morling N. 2006. A multiplex assay with 52 single nucleotide polymorphisms for human identification. Electrophoresis. 27, 1713–1724.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Philips C., Fang R., Ballard D. Fondevila M., Harrison C., Hyland F., Musgrave-Brown E., Proff C., Ramos-Luis E., Sobrino B., Carracedo A., Furtado M.R., Syndercombe- Court D., Schneider P.M.; SNPforID Consortium. 2007. Evaluation of the Genplex SNP typing system and a 49plex forensic marker panel. Forensic Sci. Int. Genetics. 1, 180–185.

    Article  Google Scholar 

  36. 36.

    Pakstis A.J., Speed W.C., Fang R. Hyland F.C.L, Furtado M.R, Kidd J.R, Kidd K.K. 2010. SNPs for a universal individual identification panel. Hum. Genet. 127, 315–324.

    Article  PubMed  Google Scholar 

  37. 37.

    Kidd K.K. 2011). Population Genetics of SNPs for Forensic Purposes (Updated). NIJ Final Report.

  38. 38.

    Musgrave-Brown E., Ballard D., Balogh K., Bender K., Berger B., Bogus M., Børsting C., Brion M., Fondevila M., Harrison C., Oguzturun C., Parson W., Phillips C., Proff C., Ramos-Luis E., et al. 2007. Forensic validation of the SNPforID 52plex assay. Forensic Sci. Int. Genet. 1, 186–90.

    Article  PubMed  Google Scholar 

  39. 39.

    Agena Bioscience. 2014). iPLEX Pro Sample ID Brochure.

  40. 40.

    Johansen P., Andersen J.D., Borsting C., Morling N. 2013. Evaluation of the iPLEX Sample ID Plus Panel designed for the Sequenom MassARRAY system. A SNP typing assay developed for human identification and sample tracking based on the SNPforID panel. Forensic Sci. Int. Genet. 7, 482–487.

    CAS  PubMed  Google Scholar 

  41. 41.

    Life Technologies. 2014). HID-Ion AmpliSeq Identity Panel Data Sheet. 2 P.

  42. 42.

    Illumina. 2014). ForenSeq DNA Signature Prep Kit Data Sheet.

  43. 43.

    Li Y., Liu Y., Lin Y. 2015. Typing of 67 SNP loci on X chromosome by PCR and MALDI-TOF MS.Res. Genet. 2015, ID 374688.

  44. 44.

    Promega. 2013). PowerPlex® Fusion Systems Data Sheet.

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Correspondence to V. A. Stepanov.

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Original Russian Text © V.A. Stepanov, K.V. Vagaitseva, V.N. Kharkov, A.A. Cherednichenko, A.V. Bocharova, 2016, published in Molekulyarnaya Biologiya, 2016, Vol. 50, No. 3, pp. 445–456.

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Stepanov, V.A., Vagaitseva, K.V., Kharkov, V.N. et al. Panel of X-linked single-nucleotide polymorphic markers for DNA identification (XSNPid) based on multiplex genotyping by multilocus PCR and MALDI-TOF mass spectrometry. Mol Biol 50, 387–397 (2016).

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  • single nucleotide polymorphic markers (SNPs)
  • X chromosome
  • MALDI-TOF mass spectrometry
  • multiplex genotyping
  • DNA identification
  • population genetics
  • genetic diversity
  • human populations