Name: Real-time Tracking of DNA Fragment Separation by Smartphone (Video) | JoVE Journal
Uploaded: 2017-06-01T15:00:00.000+00:00
Duration: 6 min 58 s
Description: Learn SNAP gel electrophoresis for real-time DNA fragment separation using a smartphone. This method reduces chemical consumption and experiment time while enabling efficient visualization of DNA migration.

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5 minutes

We gratefully acknowledge support from National Natural Science Foundation of China (No. 21205078) and the Research Fund for the Doctoral Program of Higher Education of China (No.20123120110002). This work was partially supported by the National Key Research and Development Program of China (2016YFB1102303), the National Basic Research Program of China (973Program; 2015CB352001), and the National Natural Science Foundation of China (61378060).

1. Basic Design of the SGE Chip
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Based on the SGE design, fabricate combs to create the wells in the chip for loading the DNA sample.</li>
</ol>
2. Preparation of the Agarose Gel
<ol>
	<li>Prepare 0.5&#215; TBE by mixing 10&#215; TBE (1&#215; TBE = 89 mM Tris, 89 mM boric acid, and 2 mM EDTA; pH 8.4) and distilled water in a 1:19 ratio.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Remove the comb from the gels and add 0.9 mL of 0.5x TBE buffer to cover the gel.</li>
</ol>
3. Running the Electrophoresis in the SGE Chip
<ol>
	<li>Turn on the LED light source and place a 425 to 505 nm bandpass filter above the light source.</li>
	<li>Mix 14.4 &#181;L of a 100 bp DNA ladder and 1.6 &#181;L of SYBR Green using a vortexer.</li>
	<li>Load 4 &#181;L of mixture into each well of the agarose gel in the SGE chip (Figure 2).</li>
	<li>Put the electrode into the two lanes of the SGE chip.</li>
	<li>Place a 550 to 700 nm bandpass filter above the SGE chip.</li>
	<li>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).</li>
	<li>When the electrophoresis is finished 10 min later, turn off the power source and the LED light.</li>
	<li>Clean the SGE chip.</li>
</ol>

<ol><li>Carle, G. F., Olson, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+of+chromosomal+DNA+molecules+from+yeast+by+orthogonal-field-alternation+gel+electrophoresis%5BTitle%5D)+AND+%22Nucleic.+Acids.+Res%22%5BJournal%5D)">Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis.</a> Nucleic. Acids. Res. 12 (14), 5647-5664 (1984).</li>
<li>McDonell, M. W., Simon, M. N., Studier, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+restriction+fragments+of+T7+DNA+and+determination+of+molecular+weights+by+electrophoresis+in+neutral+and+alkaline+gels%5BTitle%5D)+AND+%22J.+Mol.+Biol%22%5BJournal%5D)">Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels.</a> J. Mol. Biol. 110 (1), 119-146 (1977).</li>
<li>Fangman, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+of+very+large+DNA+molecules+by+gel+electrophoresis%5BTitle%5D)+AND+%22Nucleic.+Acids.+Res%22%5BJournal%5D)">Separation of very large DNA molecules by gel electrophoresis.</a> Nucleic. Acids. Res. 5 (3), 653-665 (1978).</li>
<li>Blum, H., Beier, H., Gross, H. J. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/elps.1150080203/abstract">Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels.</a> Electrophoresis. 8 (2), 93-99 (1987).</li>
<li>Gödde, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophoresis+of+DNA+in+human+genetic+diagnostics-state-of-the-art%2C+alternatives+and+future+prospects%5BTitle%5D)+AND+%22Electrophoresis%22%5BJournal%5D)">Electrophoresis of DNA in human genetic diagnostics-state-of-the-art, alternatives and future prospects.</a> Electrophoresis. 27 (5-6), 939-946 (2006).</li>
<li>Studier, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+bacteriophage+T7+early+RNAs+and+proteins+on+slab+gels%5BTitle%5D)+AND+%22J.+Mol.+Biol%22%5BJournal%5D)">Analysis of bacteriophage T7 early RNAs and proteins on slab gels.</a> J. Mol. Biol. 79 (2), 237-248 (1973).</li>
<li>Sugden, B., De Troy, B., Roberts, R. J., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agarose+slab-gel+electrophoresis+equipment%5BTitle%5D)+AND+%22Anal.+Biochem%22%5BJournal%5D)">Agarose slab-gel electrophoresis equipment.</a> Anal. Biochem. 68 (1), 36-46 (1975).</li>
<li>Rabilloud, T., Chevallet, M., Luche, S., Lelong, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-dimensional+gel+electrophoresis+in+proteomics%3A+Past%2C+present+and+future%5BTitle%5D)+AND+%22J.+Proteomics%22%5BJournal%5D)">Two-dimensional gel electrophoresis in proteomics: Past, present and future.</a> J. Proteomics. 73 (11), 2064-2077 (2010).</li>
<li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Agarose gel electrophoresis for the separation of DNA fragments.</a> J. Vis. Exp. (62), e3923(2012).</li>
<li>Gasser, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-strand+conformation+polymorphism+%28SSCP%29+for+the+analysis+of+genetic+variation%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Single-strand conformation polymorphism (SSCP) for the analysis of genetic variation.</a> Nat. Protoc. 1 (6), 3121-3128 (2006).</li>
<li>Matselyukh, B. P., Yarmoluk, S. M., Matselyukh, A. B., Kovalska, V. B., Kocheshev, I. O., Kryvorotenko, D. V., Lukashov, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+of+cyanine+dyes+with+nucleic+acids%3A+XXXI.+Using+of+polymethine+cyanine+dyes+for+the+visualization+of+DNA+in+agarose+gels%5BTitle%5D)+AND+%22J.+Biochem.+Biophys.+Methods%22%5BJournal%5D)">Interaction of cyanine dyes with nucleic acids: XXXI. Using of polymethine cyanine dyes for the visualization of DNA in agarose gels.</a> J. Biochem. Biophys. Methods. 57 (1), 35-43 (2003).</li>
<li>Lunn, G., Sansone, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ethidium+bromide%3A+destruction+and+decontamination+of+solutions%5BTitle%5D)+AND+%22Anal.+Biochem%22%5BJournal%5D)">Ethidium bromide: destruction and decontamination of solutions.</a> Anal. Biochem. 162 (2), 453-458 (1987).</li>
<li>Li, Z., Liu, C., Zhang, D., Luo, S., Yamaguchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Capillary+electrophoresis+of+RNA+in+hydroxyethylcellulose+polymer+with+various+molecular+weights%5BTitle%5D)+AND+%22J.+Chormatogr.+B%22%5BJournal%5D)">Capillary electrophoresis of RNA in hydroxyethylcellulose polymer with various molecular weights.</a> J. Chormatogr. B. 1011, 114-120 (2016).</li>
<li>Li, Z., Liu, C., Ma, S., Zhang, D., Yamaguchi, Y. <a target="_blank" href="http://pubs.rsc.org/en/Content/ArticleLanding/2016/AY/C5AY02705E#!divAbstract">Analysis of the inhibition of nucleic acid dyes on polymerase chain reaction by capillary electrophoresis.</a> Anal. Methods-UK. 8 (11), 2330-2334 (2016).</li>
<li>Liu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developing+a+fluorescence-coupled+capillary+electrophoresis+based+method+to+probe+interactions+between+QDs+and+colorectal+cancer+targeting+peptides%5BTitle%5D)+AND+%22Electrophoresis%22%5BJournal%5D)">Developing a fluorescence-coupled capillary electrophoresis based method to probe interactions between QDs and colorectal cancer targeting peptides.</a> Electrophoresis. 37 (15-16), 2170-2174 (2016).</li>
<li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+capillary+electrophoresis+method+to+explore+the+self-assembly+of+a+novel+polypeptide+ligand+with+quantum+dots%5BTitle%5D)+AND+%22Electrophoresis%22%5BJournal%5D)">A capillary electrophoresis method to explore the self-assembly of a novel polypeptide ligand with quantum dots.</a> Electrophoresis. 37 (15-16), 2156-2162 (2016).</li>
<li>Miao, P., et al. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0013468616300342">Nuclease assisted target recycling and spherical nucleic acids gold nanoparticles recruitment for ultrasensitive detection of microRNA.</a> Electrochim. Acta. 190, 396-401 (2016).</li>
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No conflicts of interest are declared.

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...

Slab gel electrophoresis (SGE) is the most effective method for DNA fragment separation1,2,3,4,5 and thus it is deemed a versatile tool in biochemical and biological analyses6,7,8. 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 SGE9,10, is mutagenic and cancerogenic11,12. Thus, gloves should always be worn when handing gels containing EtBr.
Capillary electrophoresis (CE) has numerous advantages13,14,15,16,17 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 (&#60;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.

<table><tbody><tr><td>10&amp;times;TBE</td><td>Beijing Solarbio Science &amp;amp; Technology Co., Ltd.</td><td>T1051</td><td></td></tr><tr><td>50 bp DNA ladder</td><td>Takara Bio Inc.</td><td>3421A</td><td></td></tr><tr><td>100 bp DNA ladder</td><td>Takara Bio Inc.</td><td>3422A</td><td></td></tr><tr><td>1 kbp DNA ladder</td><td>Takara Bio Inc.</td><td>3426A</td><td></td></tr><tr><td>SYBR GREEN</td><td>Takara Bio Inc.</td><td>5760A</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich Corporate</td><td>V900510</td><td></td></tr></tbody></table>

real-time tracking of dna fragment separation by smartphone

Real-time Tracking of DNA Fragment Separation by Smartphone

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.

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 ...

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.

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 ...

The passage introduces a new system about SNAP gel electrophoresis. This system can not only greatly reduce the chemical conception experiment time, but also can realize real-time tracking in a separation in the agarose gel by smartphone. Those three pictures represent a typical result after gel electrophoresis of 50, 100, and 1, 000 base pairs in a ladder.Each DNA fragment was well separated. SNAP gel electrophoresis is the most inventive way for DNA fragment separation, and, thus, it is deemed a vessel tutor in biochemical and biological analysis. This time I will show you a new method about SNAP gel electrophoresis experiment.Okay, let's begin. First, we weigh out 0.1 gram of agarose gel and put it into a flask, a TPE buffer into the flask. Volume of the prepared solution should be not more than one third of the capacity of the flask.Seal the flask with preservative fume, and then the agarose buffer mixture was heated by a microwave. Prior to putting the flask into microwave, we make some holes in the preservative fume in case of explosion. Hold the mounted agarose solution into the channels in the SNAP gel electrophoresis tube.Lay the comb into the solution. Mounted solution will cool down and become gel state at room temperature some minutes later. Take out of the comb to create wells and add TBE Buffer to.We have constructed a simple real time tracking DNA fragment separation system to observe the motion state of DNA fragments by smartphone. First mix the DNA ladder with SYBR Green One by centrifuge. Load the mixture into the well in the agarose gel.Second, turn on the LED light source and place a band-pass filter above the LED then put the electrode into the two lanes of the chip. Next, put other band-pass filter above it. Wait on the power source.Set the electric wattage at 180 watt. Turn on smartphone to record the DNA fragment separation process. For example we use smartphone to track separation of 50 base pairs DNA ladder.SNAP gel electrophoresis is the most effective way for DNA fragment separation. However, abandoned experiments indicate that SNAP gel electrophoresis was restricted by the full length for problems. One:the separations take many hours and even days.Two:the chemical perception is very high. Three:as it requires a lot of apparatus. Four:the gel imaging system can only observe the separated DNA fragments when the experiment is finished.Furthermore, Iridium and Bromine which is commonly used in SNAP gel electrophoresis is mutagenic and cancerogenic. After the experiment, each DNA fragment was well separated. Furthermore we have separated the same samples in the four channels of the SNAP gel electrophoresis chip and the issue that the DNA fragments was the same size moved the same distance in each experiment.From the picture, DNA fragments was separated well although there were doubting DNA fragments looks white in figure B.It can be improved by optimizing the parameters of the chip and the concentration of the agarose gel. After watching this video, you must see our new message about to SNAP gel electrophoresis. Also the resulting DNA fragments looks white.This can be improved by optimizing the parameters of your SNAP gel electrophoresis chip and the concentration of the agarose gel. What important point is, that you must be very careful during intakes paramount because it needs high voltage and it can become reagent.

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Learn SNAP gel electrophoresis for real-time DNA fragment separation using a smartphone. This method reduces chemical consumption and experiment time while enabling efficient visualization of DNA migration.

Real-time Tracking of DNA Fragment Separation by Smartphone

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