Short communicationHighly sensitive DNA detection with a combination of 2 DNA-intercalating dyes for microchip electrophoresis
Introduction
DNA analysis by agarose electrophoresis is one of the most basic methods in molecular biology. In the agarose gel electrophoresis, a run on the agarose gel requires 30–60 min and requires 10–100 ng amounts of DNA samples. After the electrophoresis, a separate step of imaging by densitometric scanning of a photograph or CCD image of the stained gel is necessary [1]. Ethidium bromide (EtBr) is frequently employed for DNA staining for the purpose of DNA sizing and semiquantification. Recently, some other fluorescent dyes such as SYBR Green I (SG I) and SYBR Green II (SG II) have been used for highly sensitive nucleic acid detection instead of EtBr [2].
Microchip electrophoresis has recently attracted much attention for DNA analysis due to its high efficiency, high throughput, time-saving ability, and cheap reagents [3]. For the commercial instrument, Hitachi SV1100, a loading gel containing EtBr is used; and the lower detection limit of DNA is 0.1 ng/μl. In the present study, we examined the DNA detection limit of a Hitachi SV1100 and an EtBr and SG II mixture instead of only EtBr. The accuracies of DNA sizing and quantification were also examined.
Section snippets
Sample preparation and reagents
Sample DNA fragments of 322 bp derived from pUC118 (3162 bp) were digested with PvuII (TOYOBO, Tokyo, Japan), separated by gel electrophoresis on agarose, and then purified with a QIAquick Gel Extraction Kit (QIAGEN, Tokyo, Japan). PCR markers (50 bp, 150 bp, 300 bp, 500 bp, and 766 bp) and a low-molecular-weight DNA ladder (25 bp, 50 bp, 75 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 500 bp, and 766 bp) were purchased from NEB (Ipswich, MA). EtBr, SG I, and II (concentration not given) were obtained
Results and discussion
The 322-bp DNA fragment was analyzed to evaluate the ability of the Hitachi SV1100 with the i-DNA 12 kit to generate consistent results with respect to DNA sizing and quantification like the original loading gel in the i-chip DNA kit. The Hitachi SV1100 is capable of estimating DNA size between 100 and 800 bp with the original loading gel [3]. The concentration of the internal control DNA fragments measured manually was 2.0 ng/μl, and we used 20 ng/μl. A single peak corresponding to 322-bp DNA was
References (6)
- et al.
Letter: unwinding the DNA helix by intercalation
J. Mol. Biol.
(1973) - et al.
Molecular Cloning: A Laboratory Manual
(2001) The Handbook, A Guide to Fluorescent Probes and Labeling Technologies
(2005)
Cited by (10)
Highly sensitive detection of MUC1 by microchip electrophoresis combining with target recycling amplification and strand displacement amplification
2022, Journal of Pharmaceutical and Biomedical AnalysisCitation Excerpt :Compared with other methods, the merits of MCE are small size, high-throughput, rapid and high-resolution separation, convenient operation and the small sample/reagent consumption [12]. MCE has been widely exploited for the analysis of biological samples, such as bacteria [13], proteins [14], nucleic acids [15] and antibiotics [16]. However, the sensitivity of MCE is limited due to its narrow separation channel and low sample volume.
Robust and easy-to-use microchip electrophoresis within sub-millimeter channels for fast and highly efficient separation
2021, TalantaCitation Excerpt :In the inset in Fig. S7B, we fitted the data points for a 200 bp, low concentration DNA fragment (2.2, 4.3, and 8.7 ng mL−1) with the intercept set to 0 and obtained a slope with a coefficient of 6.49 × 10−4 (R2 = 0.999). The limit of detection (LOD) of the 200 bp DNA fragment was found to be 2.3 ng mL−1, which is better than the previously reported LOD [42,43]. The LOD was calculated by considering three times the noise of the baseline.
A microchip electrophoresis-based assay for ratiometric detection of kanamycin by R-shape probe and exonuclease-assisted signal amplification
2018, TalantaCitation Excerpt :Thus, it has been widely adopted in biochemical analysis and can be acted as a candidate technique for developing screening method [12,13]. In recent years, there are some reports for antibiotics detection using MCE [14–16]. For example, Henry et al. successfully separated within 1 min penicillin or ampicillin by MCE with pulsed amperometric detection, and its detection limit reached to 5 µM [17].
Fast high-throughput screening of angiotensin-converting enzyme insertion/deletion polymorphism by variable programmed electric field strength-based microchip electrophoresis
2016, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesCitation Excerpt :Moreover, ME techniques allow DNA analysis using sub-microliter (<μL) volumes and high electric fields with on-capillary detection leading to shorter analysis time, higher resolution, and greater precision than SGE, which commonly needs 2–3 h of analysis time. Thus, the application of ME to biological research on DNA [18–21], proteins [22,23], neurotransmitters [24–26], and nerve agent detections [27] has been widely reported. In particular, since programmed electric field strength (PEFS) in ME is a fast and simple-to-use analysis technique without loss of resolving power by changing the electric field for analysis of target DNA molecules with a specific length, many applications of PEFS have been reported [28–35].
Recent applications of microchip electrophoresis to biomedical analysis
2015, Journal of Pharmaceutical and Biomedical AnalysisPhotoelectrochemical measurement of Epstein-Barr virus DNA in peripheral blood serum of nasopharyngeal carcinoma patients
2019, AIP Conference Proceedings
- 1
Authors were equal contributors to this work.