Development of highly sensitive and low-cost DNA agarose gel electrophoresis detection systems, and evaluation of non-mutagenic and loading dye-type DNA-staining reagents.

Highly sensitive and low-cost DNA agarose gel detection systems were developed using non-mutagenic and loading dye-type DNA-staining reagents. The DNA detection system that used Midori Green Direct and Safelook Load-Green, both with an optimum excitation wavelength at ~490 nm, could detect DNA-fragments at the same sensitivity to that of the UV (312 nm)-transilluminator system combined with ethidium bromide, after it was excited by a combination of cyan LED light and a shortpass filter (510 nm). The cyan LED system can be also applied to SYBR Safe that is widely used as a non-toxic dye for post-DNA-staining. Another DNA-detection system excited by black light was also developed. Black light used in this system had a peak emission at 360 nm and caused less damage to DNA due to lower energy of UV rays with longer wavelength when compared to those of short UV rays. Moreover, hardware costs of the black light system were ~$100, less than 1/10 of the commercially available UV (365 nm) transilluminator (>$1,000). EZ-Vision and Safelook Load-White can be used as non-mutagenic and loading dye-type DNA-staining reagents in this system. The black light system had a greater detection sensitivity for DNA fragments stained by EZ-Vision and Safelook Load-White compared with the commercially available imaging system using UV (365 nm) transilluminator.

Introduction

DNA separation and detection by agarose gel electrophoresis is one of the most frequently used techniques in life sciences [1-3]. Traditionally, DNA fragments loaded on agarose gels have been stained with ethidium bromide and detected by ultraviolet (UV)-transilluminator system [1, 4–7]. This system is a highly sensitive and low-running-cost method that has been used by many molecular biology researchers to visualize DNA in agarose gel after electrophoresis [8-11]. However, ethidium bromide and UV-light system require careful handling of the staining solution and short wavelength UV (312 nm)-transilluminator because of the mutagenic effects of ethidium bromide and high energy of short-wavelength UV light that are harmful to users [12, 13]. Moreover, DNA strands are damaged by high-energy short-wave UV rays [14]. Non-mutagenic alternative DNA-staining reagents, such as SYBR-Green and SYBR-Gold, have been developed to eliminate the disadvantages of DNA staining with ethidium bromide [15-20].

Recently, non-mutagenic and loading dye-type DNA-staining reagents that are simply mixed with sample DNA solutions have been developed and made available by several suppliers, such as EZ-vision (VWR Life Science, Radnor, PA, USA), Midori Green Direct (Nippon Genetics, Tokyo, Japan), Novel Juice (Bio-Helix, Keelung City, Taiwan) and Safelook-series (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan). These DNA-staining reagents are used in small amounts, thereby bearing low cost when compared with other post-staining types or precast-gel type reagents such as SYBR series, and, owing to their low toxicity, they can be used safely to detect DNA fragments on agarose gels after electrophoresis [21-26]. However, the use of these novel non-mutagenic DNA-staining reagents requires an additional excitation light and optical filter system to acquire high detection sensitivity for DNA fragments; these reagents are excited by wavelengths that are longer than the short wavelengths of the UV light used with ethidium bromide DNA staining. Moreover, detection sensitivity of these non-mutagenic DNA-staining reagents is lower than that of ethidium bromide-UV transilluminator system, and excitation by longer UV wavelengths employed with those novel reagents causes less damage to DNA strands [18]. Here, two novel, simple, and low-cost DNA detection systems, using non-mutagenic and loading dye-type of DNA-staining reagents, to detect DNA fragments by agarose gel electrophoresis were developed. In addition, sensitivities of loading dye-type DNA-staining reagents were evaluated in optimized DNA-detection systems using the excitation-light systems.

Materials and methods

Materials

Midori Green Direct (Nippon Genetics), Novel Juice (Bio-Helix), Safelook Load-Green (Fujifilm Wako Pure Chemical Corporation), EZ-Vision One (VWR Life Science), and Safelook Load-White (Fujifilm Wako Pure Chemical Corporation) were used as loading dye-type of DNA-staining reagents. Midori Green Direct was supplied as 10× loading dye. Novel Juice, Safelook Load-Green, EZ-Vision One, and Safelook Load-White were supplied as 6× loading dye. SYBR Safe (Thermo Fisher Scientific, Carlsbad, CA) was used as a major nontoxic post-DNA-staining reagent. A 1Kb DNA Ladder RTU (Bio-Helix, 100 ng/μL) was used as DNA ladder marker (10, 8.0, 6.0, 5.0, 4.0, 3.0, 2.5, 2.0, 1.5, 1.0, 0.75, 0.50, 0.25 kbp). Agarose S (Nippon Gene, Tokyo, Japan) was used for DNA gel electrophoresis.

DNA agarose gel electrophoresis

DNA agarose gel electrophoresis was performed for 25 min at 150 V with 0.8% agarose gel in a buffer containing 89 mM Tris, 89 mM borate, and 2 mM EDTA (TBE) [1, 7].

Excitation light and filter system

Compact LED viewer (https://www.sanplatec.co.jp/product_pages.asp?arg_product_id=SAN26955, Sanplatec, Osaka, Japan) as blue LED light (470 nm) and cyan LED stand light (https://item.rakuten.co.jp/holkin-shop/hlk-12led-490nm-495nm/, hlk-12led-490nm-495nm; Holkin, Gifu, Japan) as cyan LED light (490–495 nm) were used to excite Midori Green Direct, Novel Juice, and Safelook Load-Green. Compact black light blue lamp 27 W (https://item.rakuten.co.jp/denzaido/4992712060051/, 360 nm, FPL27BLB; Sankyo Denki, Tokyo, Japan) was used as black light to excite EZ-Vision and Safelook Load-White. A shortpass filter (https://www.asahi-spectra.co.jp/asp/syousaik.asp?key1=f41&key2=SV0510, 50 × 50 mm, SV0510; Asahi Spectra, Tokyo, Japan) was used as an excitation light filter for the cyan LED system.

Emission filters

The common filters for photography, SC-42 (420 nm), SC-46 (460 nm), SC-48 (480 nm), SC-52 (520 nm), SC-54 (540 nm), and SC-56 (560 nm) (75 × 75 mm; Fujifilm, Tokyo, Japan) were used as longpass filters for emission systems.

Commercially available imaging systems for DNA agarose gel electrophoresis

STAGE-One (AMZ System Science, Osaka, Japan) was used to visualize DNA ladder markers with ethidium bromide by UV-transilluminator system (312 nm excitation). STAGE-2000 (AMZ System Science) was used to visualize DNA ladder markers with EZ-vision and Safelook Load-White by UV-transilluminator system (365 nm excitation) as a control of commercially available UV-transilluminator system for EZ-vision and Safelook Load-White. Blue-LED transilluminator BlookTM (Bio-Helix) was used to visualize DNA ladder markers with Midori Green Direct as a control of commercially available blue-LED transilluminator system for Midori Green Direct.

Photograph system

Images of DNA agarose gels after electrophoresis were recorded with a digital camera PowerShot G12 (Canon, Tokyo, Japan) mounted on a photo stand shaded by blackout curtain. None of the images were cropped, and altered by image processing methods such as contrasting and enhancement.

Determination of detection sensitivities by successive dilutions of DNA markers on agarose gels

Fluorescent intensity of successive dilutions of DNA markers on agarose gels was analyzed for each sample by “Plot Profile” of Image-J [27]. Detected sensitivity was defined by the dilution when all the peaks of 13 DNA marker fragments were detected. The detection limit at standard volume, 1/3–1/6 volume, and <1/10 volume of DNA markers was defined as moderate, moderate-high, and high, respectively. When a part of the 13 peaks could not be detected with standard volume (5 μL), the sensitivity was defined as low.

Results and discussion

Excitation and emission of non-mutagenic and loading dye-type DNA-staining reagents

Non-mutagenic and loading dye-type DNA-staining reagents used in this study were classified according to their excitation wavelength required for detection of DNA fragments in agarose gel after electrophoresis (Table 1). Midori Green Direct, Novel Juice, and Safelook Load-Green were excited by blue (470 nm) or cyan (490–495 nm) light and showed green fluorescence. EZ-Vision and Safelook Load-White were excited by ultraviolet-A (~365 nm) and they release blue fluorescence. In contrast, ethidium bromide was well-excited by short-wave UV rays (312 nm) with high energy. Detection limit of the ethidium bromide staining was evaluated by successive dilutions of DNA markers (standard volume to 1/30 volume) (Fig 1A). The detection limit of staining reagents was defined by the dilution when all peaks of 13 DNA maker fragments were detected, as described in materials and methods (Fig 1 and S1 Fig). DNA markers stained with ethidium bromide could be detected by using 1/10 of standard DNA marker volume (500 ng, 5 μL). This result indicated high sensitivity of ethidium bromide DNA staining. In this study, two DNA detection systems for agarose gel electrophoresis were built by combining a common, commercially available LED or black light (S2 Fig) and optical filters for photography, using loading dye-type DNA-staining reagents. Schematic diagram of these DNA detection systems and photographs of the systems are shown in Fig 2 and S3 Fig, respectively.

Table 1

Excitation and emission wavelengths of non-mutagenic and loading dye-type DNA-staining reagents.

Excitation light	DNA-staining reagent*1	Excitation wavelength (nm)*2	Emission wavelength (nm)*2
blue or	Midori Green Direct	490	530
cyan LED	Novel Juice	495	537
	Safelook Load-Green	490	525
	SYBR Safe*3	502	530
UV-A	EZ-Vision	364	454
	Safelook Load-White	370	470

*1Midori Green Direct was supplied as 10× loading dye. Novel Juice, Safelook Load-Green, EZ-Vision, and Safelook Load-White were supplied as 6× loading dye.

*2Optimum excitation and emission wavelengths for DNA-staining reagents were described in each instruction manual.

*3SYBR Safe is listed because the reagent is widely used as a non-mutagenic DNA-staining reagent, although the reagent is of a post-staining type or precast-gel type.

Fig 1

Detection of DNA ladder markers stained with loading dye-type DNA-staining reagents for excitation by blue or cyan-LED light.

(a) Detection by UV (312 nm)-transilluminator system (STAGE-One, AMZ System Science) and BlookTM (470 nm, Bio-Helix); (b) Detection by blue-LED light (470 nm) excitation. SC-54 filter was used as longpass emission filter; (c) Detection by combination of cyan LED light (490–495 nm) excitation and a shortpass filter (510 nm). SC-54 filter was used as longpass emission filter. Each DNA-staining reagent was represented by underlined letters. DNA ladder markers were loaded by successive dilution. Lane 1, standard volume (5 μL (500 ng), 1 volume); lane 2, 1/2 volume; lane 3, 1/3 volume; lane 4, 1/6 volume; lane 5, 1/10 volume; lane 6, 1/15 volume; lane 7, 1/20 volume; lane 8, 1/30 volume.

Fig 2

Schematic diagram of DNA detection system for agarose gel electrophoresis using vertical illumination system.

Shortpass excitation filter, SV0510, was used for cyan-LED light. Longpass SC-46 emission filter was used for black light system, and longpass SC-54 emission filter was used for blue- or cyan-LED light.

High-sensitivity detection system of DNA fragments by blue or cyan LED light-excited DNA-staining reagents

Midori Green Direct, Novel Juice, and Safelook Load-Green are generally excited by blue LED light system. Actually, commercially available Blook system that is equipped by blue LED (470 nm) could detect Midori Green Direct-stained DNA markers (Fig 1A). The sensitivity of Midori Green Direct-stained DNA markers was evaluated by low-cost blue LED light (Compact LED viewer [470 nm], Sanplatec) whose price was ~$180 (Fig 1B and S4A Fig). Midori Green Direct-stained DNA markers were also detected by the low-cost blue LED viewer when 1/3 volume of DNA markers was loaded on an agarose gel, although the sensitivity was slightly less than that of the Blook system, which could detect 1/6 volume of DNA markers (Fig 1A and 1B). Next, to develop a low-cost DNA detection system with higher sensitivity using non-mutagenic DNA-staining reagents, Cyan LED (490–495 nm, Holkin), which emitted the optimum wavelength to excite Midori Green Direct-stained DNA markers (Table 1), was applied to the DNA detection system for Midori Green Direct. The light emitted by cyan LED (490–495 nm) could not be eliminated in the reflected extra light by longpass emission filters alone (SC-52, SC-54, and SC-56) (S4B Fig, cyan LED) because the wavelength of cyan LED was near the emission wavelength. To eliminate longer wavelength region in reflected light of cyan LED, a shortpass excitation filter (510 nm) was added to the excitation cyan LED light (S4B and S4C Fig, cyan LED and cyan LED + Excitation filter, 510 nm). However, longer wavelength components in cyan-LED could not be eliminated even by a combination of a shortpass excitation filter (510 nm) and a longpass emission filter (520 nm) because cyan LED also contains longer wavelength components in the range of 490–495 nm (S4C Fig). The background noise could be effectively reduced by using a longpass emission filter (540 nm) (S4C Fig). The combination of cyan LED, shortpass excitation filter (510 nm), and longpass emission filter (540 nm) reduced background noise and greatly improved the detection sensitivity of Midori Green Direct-stained DNA markers (Fig 1B and 1C). The detection limit of the improved cyan LED and shortpass excitation filter system was 1/10 volume of DNA markers and in the same range as that of the ethidium bromide-UV transilluminator system (Fig 1A and 1C). The combination system of cyan LED and the shortpass excitation filter could be also applied to other DNA-staining reagents, such as Novel Juice and Safelook Load-Green. The combination system could detect 1/10 volume of DNA markers using Safelook Load-Green, but only 1/3 volume of DNA markers using Novel Juice (Fig 1C). These results showed that Midori Green Direct and Safelook Load-Green detected DNA markers with higher sensitivity compared with Novel Juice. The hardware cost of the excitation system built by combining cyan LED and a shortpass filter was ~$280, lower than the cost of the commercially available Blook system (~$730). SC-54, as a longpass emission filter, effectively reduced background noise in both the blue LED (S4A Fig) and cyan LED systems combined with the excitation filter (510 nm) (S4C Fig).

Low-cost detection system of DNA-fragments based on UV-A-excited DNA-staining reagents

The optimum excitation wavelengths for EZ-Vision and Safelook Load-White were the longer waves of UV-A (360–370 nm) (Table 1). Black light has a peak emission spectrum at 360 nm and can excite DNA markers stained with EZ-Vision and Safelook Load-White. DNA markers stained with EZ-Vision and Safelook Load-White could be detected by black light, producing the 13 peaks with standard volume of DNA markers (Fig 3), whereas the commercially available DNA imaging system with UV transilluminator at 365 nm detected the DNA markers stained by EZ-Vision and Safelook Load-White with lower sensitivity. Black light is a very low-cost lamp that can be used to emit UV rays (~$40), in contrast to the expensive hardware of UV-transilluminator (>$1,000). SC-46, as a low-cost (~$16) longpass emission-filter, was effective in reducing background noise for the black light system (S5 Fig).

Fig 3

Detection of DNA ladder markers stained with loading dye-type DNA-staining reagents by excitation with UV-A light.

(a) Detection of DNA fragments stained by EZ-Vision; (b) Detection of DNA fragments stained with Safelook Load-White. EZ-Vision and Safelook Load-White were excited by black light (~360 nm) or UV (365 nm) transilluminator system (STAGE-2000, AMZ System Science). Each excitation system is represented with boxed letters. SC-46 was used as longpass emission-filter for black light system, and the accessory filter of STAGE-2000 was used as an emission filter for UV (365 nm)-transilluminator system (STAGE-2000). DNA ladder markers were loaded by successive dilution. Lane 1, standard volume (5 μL (500 ng), 1 volume); lane 2, 1/2 volume; lane 3, 1/3 volume; lane 4, 1/6 volume; lane 5, 1/10 volume; lane 6, 1/15 volume; lane 7, 1/20 volume; lane 8, 1/30 volume.

Versatility of two DNA detection systems developled in this study

Both cyan-LED (or blue-LED) system and black light system can be applied to DNA gel extraction from agarose gel because agarose gel fits in the open space of both systems (Fig 2 and S3 Fig). During the cutting of the agarose gel to extract DNA fragments, DNA bands on the agarose gel excited by specific light can be visualized by orange or yellow spectacles, as an alternative to emission filters (SC-54 and SC-46). Orange spectacles (UVP, UVC-310 and Optocode, OG-HC) can be used as an alternative to SC-54 (orange filter) (S6A Fig), and yellow spectacles (TRUSCO, TSG-814Y) as an alternative to SC-46 (yellow filter) (S6B Fig).

SYBR Safe, which is widely used as a post-staining type or precast-gel type reagent, is also a non-toxic DNA-staining reagent, although its feature is different from that of the loading dye-type DNA-staining reagents used in this study. Excitation and emission wavelengths of SYBR Safe show similar features to those of Midori Green Direct, Novel Juice, and Safelook Load-Green (Table 1). To extend the versatility of the DNA detection system developed in this study, DNA agarose gel was stained with SYBR Safe (Fig 4). Consequently, DNA agarose gel stained with SYBR Safe was clearly detected by both the blue-LED and cyan-LED + Excitation filter systems. Similar to Midori Green Direct, Novel Juice, and Safelook Load-Green, SYBR Safe provided higher detection sensitivity of DNA on agarose gel by cyan-LED + Excitation filter system than by blue-LED alone (Fig 4 and S7 Fig). The detection sensitivities were the same as those of Midori Green Direct and Safelook Load-Green (Figs 1 and 4).

Fig 4

Detection of DNA ladder markers stained with SYBR Safe for excitation by blue or cyan-LED light.

(a) Detection by blue-LED light (470 nm) excitation. SC-54 filter was used as longpass emission filter; (b) Detection by combination of cyan LED light (490–495 nm) excitation and a shortpass filter (510 nm). SC-54 filter was used as longpass emission filter. DNA ladder markers were loaded by successive dilution. Lane 1, standard volume (5 μL (500 ng), 1 volume); lane 2, 1/2 volume; lane 3, 1/3 volume; lane 4, 1/6 volume; lane 5, 1/10 volume; lane 6, 1/15 volume; lane 7, 1/20 volume; lane 8, 1/30 volume.

Conclusion

Sensitivities and costs of the DNA detection system using non-mutagenic and loading dye-type DNA-staining reagents are summarized in Table 2. Midori Green Direct and Safelook Load-Green can be used for highly sensitive DNA-detection systems on DNA agarose gel in combination with cyan LED (490–495 nm) and shortpass excitation filter (510 nm) (Fig 1C); although the running cost of Safelook Load-Green is 1.7-fold that of Midori Green Direct. SYBR Safe, a post-staining reagent, can be also applied to the LED systems (Fig 4), but its running cost is higher than that of loading dye-type DNA-staining reagents because SYBR Safe is used as a staining reagent for post-staining or precast gel staining (Table 2). The sensitivities of the DNA detection system with cyan-LED developed in this study were comparable to a commercially available ethidium bromide-UV (312 nm) transilluminator system (Fig 1A and 1C). The comparison of the initial costs for laboratory set up revealed that hardware cost of the system that combined cyan LED (490–495 nm) and shortpass excitation filter (510 nm), including emission filter, was ~$300 and thus cheaper than the cost of Blook (~$730) and UV transilluminator (>$1,000).

Table 2

Sensitivities and costs of systems implemented in the detection of DNA fragments on agarose gel after electrophoresis.

DNA-staining reagent	Excitation system*1	Emission filter*2	Sensitivity*3	Hardware cost *4	Running cost *5
Ethidium bromide	UV (312 nm) transilluminator	600FS80*6	High	****	0.05
Midori Green Direct	Blue LED (BlookTM)	Blue LED (BlookTM)	Moderate–High	***	2.0
	Blue LED	SC-54	Moderate–High	**	2.0
	Cyan LED +Ex-filter (510 nm)	SC-54	High	**	2.0
Novel Juice	Cyan LED +Ex-filter (510 nm)	SC-54	Moderate–High	**	1.9
Safelook Load-Green	Cyan LED +Ex-filter (510 nm)	SC-54	High	**	3.3
SYBR Safe*7	Blue LED	SC-54	Moderate–High	**	12 or 21*8
	Cyan LED +Ex-filter (510 nm)	SC-54	High	**	12 or 21*8
EZ-Vision	Black light	SC-46	Moderate	*	1.0
	UV (365 nm) transilluminator	BP-5465*6	Low	****	1.0
Safelook Load-White	Black light	SC-46	Moderate	*	2.0
	UV (365 nm) transilluminator	BP-5465*6	Low	****	2.0

*1 Excitation systems are described in detail in materials and methods. A shortpass filter (50 × 50 mm, SV0510; Asahi Spectra, Tokyo, Japan) was used as excitation filter (Ex-filter, 510 nm). Commercially available UV (312 nm)-transilluminator system (STAGE-One; AMZ System Science) was used for detection of ethidium bromide-stained DNA. Commercially available BlookTM (Bio-Helix) was used for detection of Midori Green Direct-stained DNA. Commercially available UV (365 nm)-transilluminator system (STAGE-2000; AMZ System Science) was used for detection of EZ-Vision- and Safelook Load-White-stained DNA.

*2 Longpass filters SC-46 and SC-54 (Fujifilm) were used as emission filters.

*3 Sensitivities are represented by four-grade evaluation such as low, moderate, moderate-high, and high. Detected sensitivity was defined in materials and methods.

*4 Hardware costs containing excitation systems and emission filters are represented by * <$100, ** $100–300, *** $300–1,000, **** >$1,000.

*5 Running costs of DNA-staining reagents are represented as the ratio to the cost of EZ-Vision, which is assumed 1.0.

*6 These filters are accessories of UV-transilluminator systems.

*7 SYBR Safe is widely used as a nontoxic staining reagent for DNA agarose gel staining. Although SYBR Safe is a DNA-staining reagent for post-staining or precast gel staining, it was added in Table 2 to extend the versatility of the LED-illumination system developed in this study.

*8 Running cost of SYBR Safe was estimated as 21 for post-staining, or 12 for precast gel staining.

Another DNA-detection system was developed that was excited by black light (~360 nm). This system caused less damage compared with damage caused by short-wave UV rays (312 nm). EZ-Vision-stained and Safelook Load-White-stained DNA gels could be observed with a black light system. Running cost of EZ-Vision was half the cost of Midori Green Direct, and therefore EZ-Vision DNA staining was cost effective, although its sensitivity was less than that of Midori Green Direct. The hardware costs of EZ-Vision were <$100, and therefore the black light system is a cost-effective excitation system for DNA agarose gel electrophoresis.

Midori Green Direct should be used when high detection sensitivity is required, such as to detect small amounts of DNA fragments, whereas EZ-Vision can be implemented when DNA fragments are detected as part of the routine DNA work because EZ-Vision is the cheapest among non-mutagenic and loading dye-type DNA-staining reagents (Table 2).

Detection limits by successive dilutions of DNA markers.

Profiles of the detection limit under each condition were analyzed by “Plot Profile” of Image-J. Each excitation system is represented with boxed letters, and each DNA-staining reagent is represented by underlined letters. Blue arrows indicate detectable DNA bands. (a) Fig 1A left, lane 5; (b) Fig 1A right, lane 4; (c) Fig 1B left, lane 3; (d) Fig 1C left, lane 5; (e) Fig 1V middle, lane 3; (f) Fig 1V right, lane 5; (g) Fig 3A left, lane 1; (h) Fig 3A right, lane 1; (i) Fig 3B left, lane 1; (j) Fig 3B right, lane 1.

(PPTX)

Click here for additional data file.

Parts of the excitation system used in this study.

(a) Blue-LED (Compact LED viewer, Sanplatec); (b) Left, cyan-LED (hlk-12led-490nm-495nm, Holkin), right, cyan-LED with excitation filter (SV0510; Asahi Spectra); (c) left, compact black light blue lamp 27 W (360 nm, FPL27BLB; Sankyo Denki), right, black light to be mounted on a fluorescent lamp stand.

(PPTX)

Click here for additional data file.

A photograph of DNA agarose gel electrophoresis detection systems developed in this study.

Gel images were recorded with a digital camera PowerShot G12 (Canon) in a dark place shaded by blackout curtain. (a) Blue-LED system; (b) Cyan-LED + Excitation filter system; (c) Black light system.

(PPTX)

Click here for additional data file.

Evaluation of emission filters for detection of DNA markers stained with Midori Green Direct.

(a) Excitation by blue-LED light (470 nm); (b) Excitation by cyan-LED light (490–495 nm); (c) Excitation by combination of cyan LED (490–495 nm) and a shortpass filter (510 nm). SC-52, SC-54, and SC-56 filters were evaluated as longpass emission-filters. DNA ladder markers were loaded by successive dilution. Lane 1, standard volume (5 μL (500 ng), 1 volume); lane 2, 1/2 volume; lane 3, 1/3 volume; lane 4, 1/6 volume; lane 5, 1/10 volume; lane 6, 1/15 volume; lane 7, 1/20 volume; lane 8, 1/30 volume.

(PPTX)

Click here for additional data file.

Evaluation of emission filters for detection of DNA markers stained with DNA-staining reagents excited by black light system.

(a) Detection of DNA markers stained with EZ-Vision; (b) Detection of DNA markers stained with Safelook Load-White. Black light (~360 nm) was used to excite EZ-Vision and Safelook Load-White. SC-42, SC-46, and SC-48 filters were evaluated as longpass emission filters. DNA ladder markers were loaded by successive dilution. Lane 1, standard volume (5 μL (500 ng), 1 volume); lane 2, 1/2 volume; lane 3, 1/3 volume; lane 4, 1/6 volume; lane 5, 1/10 volume; lane 6, 1/15 volume; lane 7, 1/20 volume; lane 8, 1/30 volume.

(PPTX)

Click here for additional data file.

Orange and yellow spectacles used to visualize DNA while cutting it out from the agarose gel.

(a) Orange spectacles (UVP, UVC-310) can be used as an alternative to SC-54, which is an orange filter. (b) Yellow spectacles (TRUSCO, TSG-814Y) can be used as an alternative to SC-46, which is a yellow filter.

(PPTX)

Click here for additional data file.

Profiles of detection limit under each condition were analyzed by “Plot Profile” of Image-J. Each excitation system is represented with boxed letters, and DNA-staining reagent is represented by underlined letters. Blue arrows indicate detectable DNA bands. (a) Fig 4A , lane 3; (b) Fig 4B , lane 5.

(PPTX)

Click here for additional data file.

31 Jul 2019

PONE-D-19-19358

Development of highly sensitive and low-cost DNA agarose gel electrophoresis detection systems and evaluation of non-mutagenic and loading dye-type DNA-staining reagents

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Reviewer #1:

Motohashi reports the use of different illumination systems to detect DNA fragments stained with a set of six intercalating dyes and separated by agarose gel electrophoresis. The illumination systems included a conventional UV transilluminator, a Blook Blue LED system, a cyan LED system, and a blacklight transilluminator. Samples are loaded in successively lower amounts on separate lanes of the gel. Data are presented as black-and-white photographs, which were generated using a Canon Powershot camera. The images were processed using Image-J.

I believe that the article satisfies most, but not all of PLOS ONE's criteria for publication:

1. The study presents the results of primary scientific research.

2. To the best of my knowledge, the results have not been published elsewhere.

3a. Some experimental details are missing. In particular, my searchs were unsuccessful for the "Compact LED viewer [470 nm], Sanplatec, for the Cyan LED (490–495 nm, Holkin), for the SV0510 shortness filter mentioned, and for the compact blacklight lamp (FPL27BLB; Sankyo Denki) mentioned on page 4 and 6 of the manuscript. URLs would be useful.

3b. I do not understand the design of the illumination systems, and those systems are not described in sufficient detail for me to reproduce the results. Were the LEDs and black lights used in a commercial transilluminator, or were illuminators used with a homemade illuminator? A diagram of the homebuilt illuminators is required.

3c. It is striking that intercalating dyes from Molecular Probes are not included in this study. In particular, SYBR Safe is marketed as having lower toxicity than ethidium bromide and is used with high sensitivity for DNA detection in gel electrophoresis.

3d. I do not see the Image-J data used to construct Table 2. The criterion for the sensitivity values in Table 2 are arbitrary because no definition of fragment detection is given. The definition of detection limit is not standard. Statistical analyses of the data are missing.

3e. The spectrometer used to generate the excitation and emission wavelengths used in Table 1 is not described in the text. It would be useful to include the spectra in the manuscript. Alternatively, if these are literature values, the source of the information should be listed.

4. The author makes several conclusions. Some appear to be justified by the data, but one set of conclusions is not justified. The author compares the cost of a commercial transilluminator with the cost of the components used to construct the black-light illumination system. As a rule of thumb, the hardware components for a commercial instrument are ~10% of the cost of the final product; the commercial instrument has additional costs associated with design and development, regulatory approval, manufacturing, and marketing; once commercialized, the systems proposed by the author will have the similar costs as the other commercial instruments. The conclusion concerning the relative costs of the various systems must be removed from the paper.

5. Except for the missing experimental details, the manuscript is presented in an intelligible fashion. While the paper would benefit from minor copyediting, that editing is not required to the paper to be interpretable

6. The paper apparently meets ethical standards.

7. The paper presents raw data as images. The corresponding Image-J data are not presented.

Reviewer #2:

The detection system described in the paper will be of significant interest to individuals and organizations for which the cost of a conventional system is prohibitively expensive. They will be able to build their own instrument based on this paper. I recommend that the author provide more details of how the system can be constructed from commercially available parts, including an image of what it looks like, so that a person who is interested will be able to replicate it.

Reviewer #3:

This manuscript describes the development of low-cost imaging systems for detecting DNA, with the goal of minimizing DNA damage due to the illumination wavelengths. The author described the detection limits using several different DNA-binding dyes and several different illumination systems.

I have several concerns about this report:

First, the sensitivity estimation relied completely on visual perception. A more rigorous numerical method should be used. These are digital images that can be easily quantified using free software tools such as ImageJ.

Second, the author did not mention how the images were displayed and if any image contrasting or enhancement was used. Was the camera mounted inside a light-tight enclosure or setup in an open space?

Third, the lanes of all gel images in all of the figures should be labeled.

Finally, and most importantly, since the goal of this work was to minimize photo-induced DNA damage, I assume the author's ultimate goal is to excise DNA fragments from gels for cloning purposes. The author needs to demonstrate the utility of these low cost systems for visualizing and excising gel fragments. Systems that require bandpass emission filters may not enable one to look directly at the gel for manually cutting out of gel fragments. In addition, are the home-built imagers amenable to the cutting of gel fragments. Commercial transilluminators are designed to enable gel cutting.

If the author's goal is to simply document DNA gels and not enable gel cutting, then it is not clear if these low-cost alternatives offer the uniformity and repeatability of commercial imaging systems.

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23 Aug 2019

For Reviewer #1:

1) Some experimental details are missing. In particular, my searches were unsuccessful for the "Compact LED viewer [470 nm], Sanplatec, for the Cyan LED (490-495 nm, Holkin), for the SV0510 shortness filter mentioned, and for the compact black light lamp (FPL27BLB; Sankyo Denki) mentioned on page 4 and 6 of the manuscript. URLs would be useful.

OUR ANSWER:

Thank you for your suggestion. Information of blue- and cyan-LED system, black light system, and shortpass filter (SV0510) was added in materials and methods (p4, l　98-106).

2) I do not understand the design of the illumination systems, and those systems are not described in sufficient detail for me to reproduce the results. Were the LEDs and black lights used in a commercial transilluminator, or were illuminators used with a homemade illuminator? A diagram of the homebuilt illuminators is required.

OUR ANSWER:

Schematic diagram of these DNA detection systems was added as Figure 2. Moreover, photographs of several parts to build the DNA detection systems were displayed as Figure S2. In addition, several sentences were added in text (p6, l 153-157).

3) It is striking that intercalating dyes from Molecular Probes are not included in this study. In particular, SYBR Safe is marketed as having lower toxicity than ethidium bromide and is used with high sensitivity for DNA detection in gel electrophoresis.

OUR ANSWER:

Thank you for your suggestion. In this study, I developed two DNA detection system using loading dye-type DNA-staining reagents, NOT post-staining type reagents such as SYBR safe. However, SYBR safe is widely used. Therefore, I evaluated detection sensitivities of DNA agarose gel stained with SYBR safe (Figure 4, Figure S7, p2 l 29-30, p8 l 220-232, and p8 l 240-243).

4) I do not see the Image-J data used to construct Table 2. The criterion for the sensitivity values in Table 2 are arbitrary because no definition of fragment detection is given. The definition of detection limit is not standard. Statistical analyses of the data are missing.

OUR ANSWER:

Thank you for your comments. The Image-J data were shown in Figure S1 and S7.

5) The spectrometer used to generate the excitation and emission wavelengths used in Table 1 is not described in the text. It would be useful to include the spectra in the manuscript. Alternatively, if these are literature values, the source of the information should be listed.

OUR ANSWER:

Thank you for your comment. Excitation and emission wave lengths were referred to each instruction manual. The source of excitation and emission wave lengths was described in Table 1 legend.

6) The author makes several conclusions. Some appear to be justified by the data, but one set of conclusions is not justified. The author compares the cost of a commercial trans-illuminator with the cost of the components used to construct the black-light illumination system. As a rule of thumb, the hardware components for a commercial instrument are ~10% of the cost of the final product; the commercial instrument has additional costs associated with design and development, regulatory approval, manufacturing, and marketing; once commercialized, the systems proposed by the author will have the similar costs as the other commercial instruments. The conclusion concerning the relative costs of the various systems must be removed from the paper.

OUR ANSWER:

Thank you for your comments. Costs of commercially available hardware include design and development, regulatory approval, and manufacturing etc., as described by reviewer #1. Therefore, commercially available systems are prohibitively expensive, as pointed out by reviewer #2. In this study, I would like to compare initial cost for laboratory setting up. These handmade LED-systems could be set up by 1/2 to 1/3, compared with commercially available Blook system or UV-transilluminator system. Furthermore, Black light system could be set up by ~$100. I believe that cost down of these instruments is helpful for researchers in a laboratory. My final goal is not that these developed DNA detection systems is marketed, and an initial costs for laboratory set up is reduced. For this purpose, hardware cost was described in text, however, I edited this sentence to explain these contexts (p8, l 246-249). I believe that comparison of initial hardware costs between commercially system and the developed systems in this study is significant information to build their DNA detection system in a laboratory.

7) Except for the missing experimental details, the manuscript is presented in an intelligible fashion. While the paper would benefit from minor copyediting, that editing is not required to the paper to be interpretable.

OUR ANSWER:

Several figures and supplemental figures were added to explain experimental details (Figure 2 and 4, Figure S1, S2, S3, S6, and S7).

For Reviewer #2:

1) The detection system described in the paper will be of significant interest to individuals and organizations for which the cost of a conventional system is prohibitively expensive. They will be able to build their own instrument based on this paper. I recommend that the author provide more details of how the system can be constructed from commercially available parts, including an image of what it looks like, so that a person who is interested will be able to replicate it.

OUR ANSWER:

Thank you for your valuable suggestion, schematic diagram of these systems and photographs of several parts were added as Figure 2, Figure S2, S3, and S6, to build their own instruments based on this study. In addition, several sentences in text were modified and added (p5 l 124-125, p6 l 153-157, and p7 l 211-219)

For Reviewer #3:

1) The sensitivity estimation relied completely on visual perception. A more rigorous numerical method should be used. These are digital images that can be easily quantified using free software tools such as ImageJ.

OUR ANSWER:

Thank you for your valuable comments, data in this article were analyzed by “Plot Profile” of Image-J, as described in materials and methods (p5, l 128-135). To demonstrate clearly, data analyzed with Image-J were displayed as Figure S1 and S7.

2) The author did not mention how the images were displayed and if any image contrasting or enhancement was used. Was the camera mounted inside a light-tight enclosure or setup in an open space?

OUR ANSWER:

All gel images were not applied any image processing. It was mentioned in text as “None of images were not altered by image processing methods such as contrasting and enhancement.” (p5, l 125-126). Schematic diagram of developed DNA detection systems was displayed in Figure 2. Two DNA detection systems were set up in blackout curtain (Figure S3). However, if light switches can be shot down in its room, the systems can be set up in an open space.

3) The lanes of all gel images in all of the figures should be labeled.

OUR ANSWER:

Thank you for your suggestion. The lanes of all gel images were labeled.

4) Since the goal of this work was to minimize photo-induced DNA damage, I assume the author's ultimate goal is to excise DNA fragments from gels for cloning purposes. The author needs to demonstrate the utility of these low cost systems for visualizing and excising gel fragments. Systems that require bandpass emission filters may not enable one to look directly at the gel for manually cutting out of gel fragments. In addition, are the home-built imagers amenable to the cutting of gel fragments? Commercial trans-illuminators are designed to enable gel cutting.

If the author's goal is to simply document DNA gels and not enable gel cutting, then it is not clear if these low-cost alternatives offer the uniformity and repeatability of commercial imaging systems.

OUR ANSWER:

Thank you for your valuable suggestion. Excitation and emission filters do not disturb accessing to agarose gel for gel-cutting. Two developed DNA detection system can be applied to gel-cutting for DNA extraction from agarose gel, and the schematic diagram and photographs were shown (Figure 2 and Figure S3). In addition, orange or yellow spectacles can be used as emission-filters for gel-cutting (Figure S6). These facts were described in text (p7, l 211-219).

Submitted filename: ResponseToReviewers_190823.docx

Click here for additional data file.

26 Aug 2019

Development of highly sensitive and low-cost DNA agarose gel electrophoresis detection systems, and evaluation of non-mutagenic and loading dye-type DNA-staining reagents

PONE-D-19-19358R1

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28 Aug 2019

PONE-D-19-19358R1

Development of highly sensitive and low-cost DNA agarose gel electrophoresis detection systems, and evaluation of non-mutagenic and loading dye-type DNA-staining reagents

Dear Dr. MOTOHASHI:

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