Fluorescence labeling of GC-rich DNA matrix with different nucleotide derivatives for hybridization analysis on a biological microarray

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Abstract

We investigated the efficiency of labeling the GC-rich promoter region of the TERT gene in the human genome with derivatives of 5′-triphosphates of 2′-deoxyuridine (dU) and 2'-deoxycytidine (dC) containing cyanine dyes Cy5 and Cy7 as a fluorescent label. The effect of modified nucleotides on the efficiency of the polymerase chain reaction was evaluated by real-time PCR, and the extent of nucleotide incorporation into the PCR product was also determined. The efficiency of DNA matrix labeling was determined by the intensity of fluorescent signal during allele-specific hybridization on a biological microarray. The highest level of biochip cell fluorescence was observed for dU-Cy7 derivatives compared to dU- and dC-Cy5 derivatives. At the same time, in the case of GC-rich DNA matrix, the use of dC-Cy5 derivatives gave a fundamentally better result compared to dU-Cy5 derivative. Thus, modified Cy7 analogs capable of incorporation into DNA during PCR are less dependent on the GC composition of the DNA matrix and are more universal fluorescent labels for diagnostic purposes. Further application of modified Cy7 analogs in the development of laboratory-on-a-chip test systems seems to be the most promising.

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About the authors

I. O. Barinova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

V. E. Shershov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

V. O. Varachev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

S. A. Surzhikov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

I. V. Grechishnikova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

О. A. Zasedateleva

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

T. V. Nasedkina

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: tanased06@rambler.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

А. V. Chudinov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Russian Federation, ul. Vavilova 32, Moscow, 119991

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Supplementary files

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2. Fig. 1. Structure of fluorescently labeled analogues of nucleotides of the Cy5 (a) and Cy7 (b) series.

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3. Fig. 2. Efficiency of PCR in the presence of different concentrations of fluorescently labeled nucleotide analogues (a) and efficiency of incorporation into the growing DNA chain (b).

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4. Fig. 3. Results of hybridization analysis on a biochip of a sample with the C/C genotype at position –146 C>T of the TERT promoter for various fluorescently labeled nucleotide analogs at a concentration of 16 μM. (a) – Hybridization patterns for a biochip fragment containing probes –146 C and –146 T; (b) – normalized fluorescence signal from cells containing a perfect duplex; (c) – discrimination ratio (the dotted line is the threshold value equal to 1.5; the area highlighted in blue includes discrimination ratio values ​​below 1.5, at which unambiguous determination of the C/C genotype for a given point is impossible.

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