Elsevier

Biosensors and Bioelectronics

Volume 61, 15 November 2014, Pages 370-373
Biosensors and Bioelectronics

Short communication
A dual-signal amplification method for the DNA detection based on exonuclease III

https://doi.org/10.1016/j.bios.2014.05.046Get rights and content

Highlights

  • A dual-signal amplification method based on two molecular beacons was designed.

  • An ssDNA with 10 T bases at 3′-termini could resist the Exo III digestion.

  • This method can discriminate single base mismatched targets well.

Abstract

A dual-signal amplification method based on two molecular beacons was designed for human hemochromatosis (HFE) gene detection. The two probes, P1 and P2, could resist the exonuclease III (Exo III) digestion due to the 3′-termini protrusion, and could coexist stably with Exo III. In the presence of HFE targets, P1 hybridized with a HFE target to form a duplex DNA with a recessed 3′-hydroxyl termini and then partially digested by Exo III, releasing the HFE target and a residual sequence (X). This X sequence could trigger the digestion of P2 probes with 6-carboxy-fluoresceins and Black Hole Quenchers and then result in the increase of fluorescence intensity. The X sequences were more stable than HFE targets and could cyclically trigger the P2 digestion for a long time even though the HFE targets were digested by Exo III. This method improved the sensitivity and reached 4 orders of magnitude in detection limit, and showed excellent selectivity to discriminate single base mismatched targets well.

Introduction

The detection of ultralow DNA concentrations is of crucial importance in many areas such as clinical diagnosis, gene therapy, and pathogen detection (Liu et al., 2009). The traditional DNA hybridization assay between targets and probes is based on a 1:1 stoichiometric ratio, which results in low sensitivity. The signal amplification strategies make it possible that one DNA target can cyclically induce multiple probes to generate signals, achieving higher sensitivity (Scrimin and Prins, 2011). So far, a variety of amplification methods have been developed based on the hybridization chain reaction (Chen et al., 2012, Huang et al., 2011), the supersandwich type assay (Chen et al., 2011a), the strand-displacement amplification (Weizmann et al., 2006) and the catalysis by nucleases such as endonuclease (Xiao et al., 2012a, Xiao et al., 2012b, Xu et al., 2009, Zou et al., 2011), Dnase I (Cui et al., 2012), duplex-specific nuclease (Yin et al., 2012), lambda exonuclease (Hsieh et al., 2010) and exonuclease III (Exo III) (Bi et al., 2012, Cui et al., 2010, Xu et al., 2012, Zuo et al., 2010).

The enzyme-assisted amplification has been widely applied due to the advantages of easy availability, high catalytic activity and excellent specificity. Exo III can digest blunt or recessed 3′-hydroxyl termini of duplex DNA with stepwise removal of mononucleotides, but has low activity on single-stranded DNA or 3′-protruding termini of double-stranded DNA (dsDNA) (Bi et al., 2012, Cui et al., 2010, Richardson et al., 1964, Zuo et al., 2010). It does not require any specific DNA sequences for recognition, which makes it as a universal nuclease for DNA digestion. So, Exo III has been used to design amplification strategies for DNA detection based on molecular beacons (Zuo et al., 2010) and double-stranded DNA probes (Cui et al., 2010). The results show 2 orders of magnitude in sensitivity better than that without any amplification. However, these single amplification procedures only continue for 30 min which restricts the magnification. Recent researches show that Exo III has the activity of digesting single-stranded DNA (ssDNA) (Xu et al., 2012, Yang et al., 2007). The digestion of ssDNA targets is one of the main causes for the cease of amplification. In order to get higher magnification, some Exo III amplification methods are coordinated with PCR (Zuo et al., 2011) and electrochemistry (Chen et al., 2011b, Fan et al., 2012, Miranda-Castro et al., 2012, Su et al., 2011, Wu et al., 2011, Xuan et al., 2012). These methods improve the sensitivity indeed, but need sophisticated processes. An exonuclease-assisted cascaded recycling amplification is proposed based on the digestion of three molecular beacons cyclically in the presence of the DNA targets, which results in 4 orders of magnitude for the detection limit (Bi et al., 2012). However, the unstable coexistence between the molecular beacons led to high background signals and poor selectivity.

In order to overcome the deficiency of target digestion by Exo III, we developed a dual-signal amplification method for human hemochromatosis (HFE) gene detection. In this method, HFE gene could cyclically trigger the partial digestion of the P1 probe to release a residual sequence (X) that could resist the Exo III digestion and induce the P2 probe digestion for a long time. This method reached 4 orders of magnitude in detection limit and showed excellent selectivity as well.

Section snippets

Materials

Oligonucleotides were custom-synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Exonuclease III was purchased from TaKaRa BiotechnologyCo., Ltd. (Dalian,China). The buffer solution for the assay was 50 mmol/L Tris–HCl (pH 8.0) containing 5 mmol/L MgCl2 and 200 mmol/L NaCl. In the fluorescence experiment, the concentrations of P1 and P2 are 1.25 and 2.5 μmol/L, respectively. The pure water (18  cm) for solution preparation was from a Millipore autopure WR600A system (USA), and was used

The principle of strategy

In this dual-signal amplification method (Scheme 1), two molecular beacons, P1 and P2, were designed with 3′-termini protrusion for resistance to Exo III. These two probes could coexist stably which lead to low background signals. In the first step amplification, P1 hybridized with HFE target to form a duplex DNA with a recessed 3′-hydroxyl termini, triggering the partial digestion of P1 by Exo III to release a HFE target and a residual sequence (X). Because Exo III had low activity for

Conclusion

In conclusion, a dual-signal amplification strategy was designed based on two molecular beacons, P1 and P2. After inducing the DNA targets and digesting by Exo III, the P1 probes released the residual sequence X that resists the Exo III digestion. The first step amplification could accumulate some amount of the X sequences that induce P2 digestion for a long time. These two probes coexisted well without any hybridization which restricts the increase of background signals with the growth of

Acknowledgments

This research work was financially supported by the National Basic Research Program of China (2010CB732402), the National Natural Science Foundation of China (Nos. 21175112 and 2137512) and the NFFTBS (No. J1030415), which are gratefully acknowledged. Professor John Hodgkiss of The University of Hong Kong is thanked for his assistance with English.

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