The Efficiency of DNA Labeling with Near-Infrared Fluorescent Dyes
- PDF / 611,661 Bytes
- 6 Pages / 612 x 792 pts (letter) Page_size
- 97 Downloads / 227 Views
CULAR BIOPHYSICS
The Efficiency of DNA Labeling with Near-Infrared Fluorescent Dyes V. E. Shershova, A. Yu. Ikonnikovaa, V. A. Vasiliskova, S. A. Lapaa, R. A. Miftakhova, V. E. Kuznetsovaa, A. V. Chudinova, and T. V. Nasedkinaa, * a
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia *e-mail: [email protected] Received July 8, 2020; revised July 16, 2020; accepted July 17, 2020
Abstract—The efficiency of DNA labeling was assessed for 2'-deoxyuridine 5'-triphosphate (dUTP) derivatives containing the Cy7 cyanine dye as a fluorophore. Two fluorescent Cy7-labeled dUTP analogs differed in the chemical structure of the linker between the fluorophore and nucleotide moieties. The efficiency of the polymerase chain reaction (PCR) and inhibition with modified nucleotides were estimated by real-time PCR. The efficiency of labeled nucleotide incorporation in PCR products was measured by quantitative electrophoresis. The efficiency of target DNA labeling was evaluated by binding the fluorescently labeled PCR products to a microarray of oligonucleotide probes immobilized in hydrogel drops (a biochip). The nearinfrared hybridization signal was detected by digital luminescence microscopy. An increase in linker length was found to provide more efficient incorporation of the labeled nucleotide. Both of the compounds provided high sensitivity and high specificity of DNA testing via allele-specific hybridization on a biochip. Keywords: fluorescence, modified nucleotides, near-infrared cyanine dyes, real-time PCR, PCR efficiency, biological microarray, allele-specific hybridization DOI: 10.1134/S0006350920050188
INTRODUCTION Various modalities are used to investigate biologically active macromolecules. One of the modalities is carrying out a reaction on a solid support surface with micro amounts of a test substance and detecting the resulting signal [1–3]. Fluorescent dyes are broadly used to label the macromolecules involved in the test procedure and thus to visualize the results. The most common dyes fluoresce in the visible range, where substantial background fluorescence of test components may be a large problem. Background fluorescence is lower with Cy5 dyes, which fluoresce in a range of 650–670 nm [4, 5]. Still lower background fluorescence may potentially be achieved in a near infrared range of 750–1000 nm, which is also known as the biological window [6, 7]. Tricarbocyanine dyes similar to Cy7 have absorption and fluorescence maximums in the near infrared region and are of special interest among organic dyes. Cy7 analogs are sufficiently stable in the conditions of molecular biological analyses and have high molar extinction coefficients and high fluorescence quantum yields [8–10]. DNA polymerases are capable of utilizing cyanine dye-labeled 2'-deoxyuridine 5'-triphosphates (dUTPs) as substrates and incorporating fluorescently Abbreviations: PCR—polymerase chain reaction; Cy7-dUTP and Cy5-dUTP—fluorescently labeled 2'-deoxyuridine 5'-triphosphate analogs.
labeled nucleotide in DNA st
Data Loading...
