Label-free and sensitive fluorescent detection of sequence-specific single-strand DNA based on S1 nuclease cleavage effects.

Zheng Guan,Ailiang Chen,Xiaoling Jiang,Zhenzhen Lv,Guiyuan Lv,Wenhui Bai,Shuming Yang,Jinchuan Liu,Albert Jeltsch

doi:10.1371/journal.pone.0108401

Abstract

The ability to detect sequence-specific single-strand DNA (ssDNA) in complex, contaminant-ridden samples, using a fluorescent method directly without a DNA extraction and PCR step could simplify the detection of pathogens in the field and in the clinic. Here, we have demonstrated a simple label-free sensing strategy to detect ssDNA by employing its complementary ssDNA, S1 nuclease and nucleic acid fluorescent dyes. Upon clearing away redundant complementary ssDNA and possibly mismatched double strand DNA by using S1 nuclease, the fluorescent signal-to-noise ratio could be increased dramatically. It enabled the method to be adaptable to three different types of DNA fluorescent dyes and the ability to detect target ssDNA in complex, multicomponent samples, like tissue homogenate. The method can distinguish a two-base mismatch from avian influenza A (H1N1) virus. Also, it can detect the appearance of 50 pM target ssDNA in 0.5 µg·mL−1 Lambda DNA, and 50 nM target ssDNA in 5 µg·mL−1 Lambda DNA or in tissue homogenate. It is facile and cost-effective, and could be easily extended to detect other ssDNA with many common nucleic acid fluorescent dyes.

Highlights

Single strand DNA or RNA analysis plays an important role in the molecular biology, medicine and diagnosis
S1 nuclease plays an important role which degrades single-stranded nucleic acid or cleaves dsDNA at the singlestranded region caused by a nick, gap, mismatch or loop
This ability makes it feasible to cleavage the possible small single-strand DNA (ssDNA) ‘bubbles’ surrounding the mismatch site induced by the complementary ssDNA [10,11], which gives the strategy a discrimination capability of sequence mismatches

Summary

Introduction

Single strand DNA or RNA analysis plays an important role in the molecular biology, medicine and diagnosis This has driven the development of various methods to detect sequence-specific DNA or RNA with high sensitivity and specificity. Some nanomaterials including gold nanoparticles, quantum dots, carbon nanotubes and graphene were used for fluorescence assays of ssDNA and RNA [1,2,3,4,5,6,7,8]. These methods have some limitations such as base selectivity, insufficient sensitivity and high-cost, especially for sequence-specific ssDNA and RNA

Methods

Results

Conclusion