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Multiplex Assay to Evaluate the Genetic Risk of Developing Human Melanoma

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Abstract

A genotyping procedure based on single-step PCR and subsequent allele-specific hybridization on a hydrogel biochip was developed to address the polymorphisms of HERC2, OCA2, SLC24A4, SLC45A2, TYR, IRF4, MC1R, MITF, PIGU, MYH7B, NCOA6, and CDK10. Amplified gene fragments were fluorescently labeled in PCR, and fluorescent signals from biochip cells were detected to evaluate how efficiently the PCR product formed a perfect duplex with an immobilized probe. The analytical characteristics of hybridization analysis were estimated for several fluorophores with different optical spectra. Cyanine dyes fluorescing in the range of Cy5 and Cy7 were synthesized for the purpose and used as 5'-tags of universal primers in single-step PCR. A Cy7 analog fluorescing in the near infrared range was found to increase the sensitivity of hybridization analysis by producing a lower background signal in the cases where target gene amplification was low.

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Authors and Affiliations

  1. Engelhardt Institute of Molecular Genetics, Russian Academy of Sciences, 119991, Moscow, Russia

    D. O. Fesenko, I. S. Abramov, V. E. Shershov, V. E. Kuznetsova, S. A. Surzhikov, I. V. Grechishnikova, V. E. Barsky, A. V. Chudinov & T. V. Nasedkina

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  1. D. O. Fesenko
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  2. I. S. Abramov
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  3. V. E. Shershov
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  4. V. E. Kuznetsova
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  5. S. A. Surzhikov
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  6. I. V. Grechishnikova
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  7. V. E. Barsky
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  8. A. V. Chudinov
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  9. T. V. Nasedkina
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Correspondence to D. O. Fesenko.

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Translated by T. Tkacheva

Abbreviations: CDK4, cyclin-dependent kinase 4 gene; CDK10, cell division protein kinase 10 gene; CDKN2A, cyclin-dependent kinase inhibitor 2A gene; GWAS, genome-wide association study; HERC2, HECT domain and RCC1-like domain 2 gene; IRF4, interferon regulatory factor 4 gene; MC1R, melanocortin 1 receptor gene; MITF, microphthalmia-associated transcription factor gene; MYH7B, myosin heavy chain 7B gene; NCOA6, nuclear receptor coactivator 6 gene; OCA2, oculocutaneous albinism II gene; PIGU, phosphatidylinositol glycan anchor biosynthesis, class U protein gene; PTEN, phosphatase and tensin homolog gene; SLC24A4, solute carrier family 24 member 4 gene; SLC45A2, solute carrier family 45 member 2 gene; TP53, tumor protein 53 (p53) gene; TYR, tyrosinase gene.

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Fesenko, D.O., Abramov, I.S., Shershov, V.E. et al. Multiplex Assay to Evaluate the Genetic Risk of Developing Human Melanoma. Mol Biol 52, 865–871 (2018). https://doi.org/10.1134/S0026893318060079

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  • DOI: https://doi.org/10.1134/S0026893318060079

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