A New Trace Analytic Method for the Determination of Sequence-Specific DNA with Fluorescent Probes

Abstract

A new method of fluorescence spectrometry detection of single-strand DNA (ssDNA) was established by hybridizing the ssDNA with its complementary ssDNA to form double-stranded DNA (dsDNA). Our results show that the fluorescence intensity increased significantly when the nucleic acid molecular “light switch"(Ru(phen)₂dppx²⁺) or Hoechst 33258 dye interacted with dsDNA, and the fluorescence intensity also increased as the DNA concentration increased. The changing law was also studied about how the fluorescence intensity changed when the two kinds of fluorescent probes interacted with oligonucleotide of different lengths and different sequences, as well as DNA-DNA′ hybridization products. Then, the effect of the bases mismatch, varying length of DNA chain, and different DNA sequences on the fluorescence intensity were explored at the same time, by detecting the specific DNA sequence of avian influenza H1N1 virus, cauliflower mosaic virus, and hepatitis C virus. Additionally, the selectivity, linear range, and sensitivity of the two probes were compared.

Keywords:

• Fluorescence spectrum
• Fluorescent probe
• Single-strand DNA
• Trace analysis method

Acknowledgments

This work was supported by the National Science Foundation of China (21075093, 20921062), the Hubei Provincial Department of Education Research (No. B20113001), and the Key Disciplines “Applied Chemistry” (201008) of Hubei University of Education.

Notes

^a The ratios = [the fluorescence intensity of mismatched base hybridization with fluorescence probe minus blank value minus the fluorescence intensity of fluorescence probe solution hst (blank value)]: [the fluorescence intensity of completely complementary hybridization with fluorescence probe minus blank value].

^a In the equation, y is the fluorescence intensity of standard solution minus blank solution and x is the concentration of standard solution.