Real-time Tracking of DNA Fragment Separation by Smartphone

Podsumowanie

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

Streszczenie

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Wprowadzenie

Slab gel electrophoresis (SGE) is the most effective method for DNA fragment separation^1,2,3,4,5 and thus it is deemed a versatile tool in biochemical and biological analyses^6,7,8. However, many experiments indicate that SGE is restricted by the following four problems: (1) the separations take many hours, and even days; (2) the chemical consumption is very high; (3) it requires a complicated apparatus (e.g., 2D electrophoresis cell, electrophoresis power supply, and gel imaging system); (4) the gel imaging system can only observe the separated DNA fragments when the experiment is finished. Furthermore, ethidium bromide (EtBr), which is commonly used in SGE^9,10, is mutagenic and cancerogenic^11,12. Thus, gloves should always be worn when handing gels containing EtBr.

Capillary electrophoresis (CE) has numerous advantages^{13,14,15,16,17} compared to SGE, such as automatic operation, short separation time, and lower consumption. However, the CE instrument is quite expensive. Therefore, to overcome those limitations, a system has been developed (Figure 1) for the separation of DNA. Such a system can not only greatly reduce the chemical consumption and save on SGE experiment time (<8 min), but it can also perform real-time tracking of the DNA separation process in the agarose gel by smartphone. By following the procedures described in this protocol, students should be able to design and fabricate the SGE chip, prepare the agarose gel in the chip, set up a simple SGE system with a smartphone, and record the DNA migration process in the agarose gel.

Protokół

1. Basic Design of the SGE Chip

1. Use any transparent plastic, such as polymethylmethacrylate (PMMA) or polycarbonate.
   Note: The SGE chip is demonstrated in Figure 1B. The SGE chip consists of cylindrical holes for the TBE buffer, channels for DNA separation, and two lanes embedded along the holes for the electrode.
2. Fabricate arrays of SGE channels in the PMMA block using a laser engraving machine.
   Note: The geometric parameters of the chip are dependent upon the size of the DNA to be separated. For example, the optimal channel parameters for the SGE chip are 2.5 mm x 4.0 mm x 90 mm (width x depth x length) if the DNA fragment is smaller than 1,000 bp.
3. Based on the SGE design, fabricate combs to create the wells in the chip for loading the DNA sample.

2. Preparation of the Agarose Gel

1. Prepare 0.5× TBE by mixing 10× TBE (1× TBE = 89 mM Tris, 89 mM boric acid, and 2 mM EDTA; pH 8.4) and distilled water in a 1:19 ratio.
2. Prepare 1.0% agarose gel for the separation of 100-bp DNA ladders.
   Note: The concentration of agarose in 0.5x TBE buffer is dependent upon the sizes of the DNA fragments to be separated.
3. Place 0.1 g of agarose into a flask and add 10 mL of 0.5x TBE buffer.
   Note: The volume of the prepared solution should be less than 1/3 of the capacity of the flask.
4. Seal the flask with preservative film and then heat the agarose/buffer mixture in a microwave (medium, 1.0 min). Prior to putting the flask into the microwave, make some holes in the preservative film in case of an explosion.
5. Pour 2.7 mL of melted agarose solution into 4 channels of the SGE chip. Place the comb into the agarose solution to create the wells.
   Note: The melted agarose solution will cool down to the gel state at room temperature 3 min later.
6. Remove the comb from the gels and add 0.9 mL of 0.5x TBE buffer to cover the gel.

3. Running the Electrophoresis in the SGE Chip

1. Turn on the LED light source and place a 425 to 505 nm bandpass filter above the light source.
2. Mix 14.4 µL of a 100 bp DNA ladder and 1.6 µL of SYBR Green using a vortexer.
3. Load 4 µL of mixture into each well of the agarose gel in the SGE chip (Figure 2).
4. Put the electrode into the two lanes of the SGE chip.
5. Place a 550 to 700 nm bandpass filter above the SGE chip.
6. Switch on the power source. Set the electric voltage to 180 V (20 V/cm). Turn on the smartphone to record the DNA fragment separation process (Figure 3).
7. When the electrophoresis is finished 10 min later, turn off the power source and the LED light.
8. Clean the SGE chip.

Wyniki

Figure 4, Figure 5, and Figure 6 represent a typical result after the gel electrophoresis of 50, 100, and 1,000 bp DNA ladders. After the experiment, the DNA fragments were well-separated. Furthermore, the same samples were separated in the 4 channels of the SGE chip, showing that DNA fragments of the same size move the same distance in each experiment.

The separation performance of the DNA ladder can be ...

Dyskusje

Agarose gel electrophoresis is widely employed for the separation of DNA, RNA, and protein. This work proposes a new method to replace the traditional gel electrophoresis protocol. Results demonstrate that 50, 100, and 1,000 bp DNA ladders can be separated well in such a small assembled device. The great advantage of this method is that not only can it separate the nucleic acids with little chemical consumption, but it can also record the separation process. Although the DNA fragments looks wide in F...

Materiały

Name	Company	Catalog Number	Comments
10×TBE	Beijing Solarbio Science & Technology Co., Ltd.	T1051
50 bp DNA ladder	Takara Bio Inc.	3421A
100 bp DNA ladder	Takara Bio Inc.	3422A
1 kbp DNA ladder	Takara Bio Inc.	3426A
SYBR GREEN	Takara Bio Inc.	5760A
Agarose	Sigma-Aldrich Corporate	V900510