Solid- and solution-phase synthesis and application of R6G dual-labeled oligonucleotide probes

Bioorg Med Chem. 2015 Oct 15;23(20):6749-56. doi: 10.1016/j.bmc.2015.08.041. Epub 2015 Sep 1.

Abstract

A novel N-TFA-protected carboxyrhodamine 6G (R6G) phosphoramidite was synthesized for use in an automated DNA synthesis to prepare 5'-labeled oligonucleotides. Deprotection and purification conditions were optimized for 5'-labeled and dual-labeled oligonucleotide probes. As an alternative we synthesized an azide derivative of R6G for CuAAC post-synthetic oligonucleotide labeling. Dual-labeled probes obtained by both methods showed the same efficacy in a quantitative PCR assay. R6G-labeled probes demonstrated superior properties in a qPCR assay in comparison with alternative HEX, JOE and SIMA dyes due to more efficient fluorescence quenching by BHQ-1. We successfully used R6G dual-labeled probes for rotavirus genotyping.

Keywords: CuAAC; Fluorescent dye; Oligonucleotide; Phosphoramidite; Rhodamine 6G; qPCR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescence
  • Fluorescent Dyes / chemistry
  • Genotype
  • Oligonucleotide Probes / chemical synthesis*
  • Oligonucleotide Probes / chemistry*
  • Rhodamines / chemistry*
  • Rotavirus / genetics
  • Solutions

Substances

  • Fluorescent Dyes
  • Oligonucleotide Probes
  • Rhodamines
  • Solutions
  • rhodamine 6G