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  • SDS-Polyacrylamide Gel Electrophoresis at Neutral pH (NuPAGE)
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Agarose gel electrophoresis is a type of gel electrophoresis that uses agarose, a natural polysaccharide isolated from red seaweed agar, as a matrix to separate molecules or components based on their size. It is an uncomplicated, yet versatile analytical method that can accommodate both research and routine laboratory works. It is nowadays considered a conventional technique for the analysis of nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

Agarose as a matrix for gel electrophoresis

Agarose is a type of natural polysaccharide isolated from red seaweed that is used to make a gel. In gel electrophoresis, the gel is cast into a block or slab of various height, length, and thickness. Once set, the gel is saturated with or submerged in an electrophoresis running buffer, acting as a support matrix. Liquid samples are applied onto a restricted area of the gel, and electrophoretic separation occurs when an electric field is applied to the unit. The generated electric force propels the components in the samples to migrate at different rates. Components with the same characteristics are separated into distinct bands until the electric force is removed from the system. The migration pattern can be visualized when the gel is stained with various dyes that are compatible with the sample.[1,2]

As a support matrix, the gel stabilizes the pH of the buffer surrounding the sample, mitigating the convection current that is induced when the electric field is applied to the electrophoresis system. The gel can act as a separation matrix due to its molecular sieving influence.[3,4] The shape and size of the pores in-between the molecules of the gel impose frictional force onto the components in the sample. Components with higher molecular weight will be subjected to greater friction than components with lower molecular rates and will, as a result, migrate slower. Thus, the resolving power of gel electrophoresis is directly influenced by the gel composition, which can be fine-tuned to accommodate the expected range of the size of the components.[3-5]

Agarose polymers form sieving pores when gelled

Agarose is isolated from the agar of various red algae species, known as Rhodophyta. Purified agarose is a linear polymer that mainly consists of repeating units of agarobiose, which is a disaccharide of alternating chains of 1,3-linked β-D-galactose and 1,4-linked 3, 6-anhydro-α-L-galactose. Additionally, agarose backbone also contains substantial amounts of methoxyl, carboxylate, sulfate and/or pyruvate.[3,6] The presence of these substitution groups and their composition determine the purity of the agarose, which also influences its melting and gelling temperature.[4]

Agarose exists in a powdered form that can be dissolved in an aqueous solution, heated and then molded by letting the agarose solution solidify. The structure of agarose gel is developed during gelation, which influences the gel properties and the subsequent electrophoretic separation. When the agarose solution is heated, the polymer strand exists in a random coil conformation.[4] When the solution begins to cool, the coiled strands unwind, and two polymer strands form a double helix. During gelation, several double helices aggregate laterally, forming suprafibers in the process. Agarose suprafibers are composed of the “pillars” made of the double helices and their “pores”.[6]

The formation of suprafiber makes agarose gels sturdy even at low gel concentration, making agarose gel easy to handle, compared to starch or polyacrylamide gels[5,6]. The pores of the suprafibers contribute to the molecular sieving influence, which is dependent on the concentration of agarose. The higher the agarose concentration, the smaller is the pore, and the finer is the molecular sieve.[5,7] In other words, gels with high agarose in their composition are more suitable for the electrophoretic separation of molecules possessing small molecular weight than gels with low agarose content, and vice versa.[7]

Preparation and setup of agarose gel electrophoresis

Agarose gel electrophoresis can be broadly divided into the following steps:

1. Agarose gel preparation:

Agarose powder can be dissolved in an electrophoresis running buffer and heated. After heating, the agarose gel solution is poured into a mold and allowed to cool at room temperature.

For a horizontal gel system, agarose gels are cast in a casting tray, and the sample application wells are simultaneously molded at the top of the gel using a comb. For a vertical gel system, however, the heated agarose gel solution is poured in the space of a gel cassette, consisting of two glass plates clamped together with a spacer in between. The sample application wells are formed by taping a row of squares or rectangles onto one of the glass plates used in the gel cassette.[5-7]

The concentration of agarose gel is expressed as a percentage of the weight of agarose to the volume of the solution buffer. Generally, the concentration of the agarose gel used in an analysis is dependent on the expected range of the size of the separated components being analyzed.[7] Along the same line, the choice of solution used for preparing agarose gel depends on the type of samples and their components. Typically, the components in the buffer used for the preparation of agarose gel are similar to those in the electrophoresis running buffer.

2. Sample preparation and loading:

Samples used in agarose gel electrophoresis are in a liquid state. Before samples are loaded into the wells, samples are mixed with a sample loading buffer, also known as sample loading dye. Generally, sample loading buffers contain a high-density, non-reactive solution and tracking dyes. When mixed with samples, high-density, non-reactive solutions such as glycerol, sucrose and Ficoll™ solutions cause the samples to sink to the bottom of the wells instead of diffusing into the electrophoresis running buffer.[3] Xylene cyanol, bromophenol blue, cresol red and orange G are electrophoresis tracking dyes that change the color of the samples from colorless to blue, violet, red and orange, respectively. The presence of (a) tracker dye(s) in the sample loading buffer aids in sample loading and allows the migration of samples to be monitored during electrophoresis.

Apart from the samples, a size marker, otherwise known as a sample ladder, is also loaded in a separate sample well before the electrophoresis is initiated. The ladder contains fragments of known size that are of the same species as the samples. They are separated along with the samples and are used to estimate the size of the bands resulting from electrophoretic separation.[2,5]

3. Electrophoretic separation:

An agarose gel electrophoresis unit is composed of an agarose gel block or slab submerged in the electrophoresis running buffer placed in a closed electrophoresis chamber. The separation is initiated when the electric current is applied to the electrophoresis unit and is terminated when the electric current is removed.[7] As with the case for gel electrophoresis, the velocity of each component is determined by their mass and net charge and is directly proportional to the applied voltage and current.[1,5,7] Simply put, higher voltage or current will result in faster electrophoretic separation because the components in the sample will migrate faster. Nevertheless, too high voltage or current can cause the bands of the separated components to be blurry or smeared[5,7] or smily[3], or it can generate excessive heat during electrophoresis, causing damage to the agarose gel or the sample.[2,3,5]

4. Visualization and downstream analysis:

Electrophoretic separation in an agarose gel results in a migration pattern of the sample ladder and of the components in the samples. However, right after the electric field is removed from the system, only the migration pattern of the tracker dye(s) and in some cases, the sample ladder, is visible to the naked eye. To visualize the resulting bands, agarose gel is stained with dyes that are compatible with the sample, revealing the pattern of components that were separated from the sample.[2,5] The stained agarose gel can be imaged and used for downstream analysis.

For agarose gel electrophoresis of DNA and RNA samples, agarose gels are stained with an intercalating dye, which inserts itself in between the bases of the nucleic acids. Ethidium bromide or SYBR Green are examples of fluorescent intercalating dyes that emit fluorescence when they are intercalated in the backbone of the molecule and exposed to short-wave ultraviolet (UV) light.[5] The UV-exposed stained gel can be imaged for record or downstream analyses.

Applications of agarose gel electrophoresis

Agarose gel electrophoresis has been applied to accommodate several types of analyses, for example:

1. Quality control and quantification of nucleic acids

The isolation of nucleic acids constitutes one of the most important laboratory works. Oftentimes, the quality and quantity of the isolated nucleic acids are determinant to the success of the downstream processes.

Electrophoretic separation of DNA in agarose gel can reveal the integrity of the isolated DNA based on the appearance of an unblurred DNA band which has little smear. The quantity of the isolated DNA can be estimated based on the comparison with the DNA ladder.[5,7]

In the case of RNA, the integrity of the isolated RNA can be determined using denaturing or nondenaturing, high percentage agarose gel. After electrophoresis, the presence of three distinct bands on the gel represents ribosomal RNAs, which can be used as an indication that the extracted RNA is intact.[5] Similar to DNA, the quantity of the isolated RNA can be calculated based on the presence of the RNA ladder.[7]

2. Separation of DNA fragments

Agarose gel electrophoresis is suitable for the separation of small, medium and medium-large DNA fragments obtained from restriction digestion and polymerase chain reactions. Based on the range of the expected size of the separated fragments, the agarose concentration can be adjusted (Table 1). The separated DNA fragments can be recovered from the agarose gel after electrophoresis for subsequent works such as molecular cloning, making the technique especially practical.[5]

Table 1 The concentration of agarose gel and the suitable range of linear DNA fragments[8]

Agarose gel concentration

(%(w/v))

Effective range of linear DNA fragments (bp)
0.5 30,000 to 1,000
0.7 12,000 to 800
1.0 10,000 to 500
1.2 7,000 to 400
1.5 3,000 to 200
2.0 2,000 to 100
3.0 1,000 to 25

In conclusion

All in all, the strength of agarose gels and the simplicity in setting up electrophoresis contribute to the popularity of the technique in research and routine analysis. The properties of agarose which allows it to accommodate samples of various ranges of molecular sizes have made agarose gel electrophoresis one of the essential techniques in biotechnology.

References

  1. Jorgenson, J. W. (1986). Electrophoresis. Analytical Chemistry, 58(7), 743A-760A. https://doi.org/10.1021/ac00298a001
  2. Westermeier, R., Gronau, S., Becket, P., Buelles, J., Schickle, H., & Theßeling, G. (2005). Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations (4th, revised ed.). Wiley-VCH Verlag.
  3. Barril, P., & Nates, S. (2012). Introduction to Agarose and Polyacrylamide Gel Electrophoresis Matrices with Respect to Their Detection Sensitivities. In S. Magdeldin (Ed.), Gel electrophoresis: Principles and basics. Rijeka, Croatia: InTech.
  4. Stellwagen, N. C., & Stellwagen, E. (2009). Effect of the matrix on DNA electrophoretic mobility. Journal of Chromatography A, 1216(10), 1917–1929. https://doi.org/10.1016/j.chroma.2008.11.090
  5. Walker, J. M. (2010). 10 Electrophoretic techniques. In K. Wilson & J. M. Walker (Eds.), Principles and Techniques of Biochemistry and Molecular Biology (7th ed.). Cambridge: Cambridge University Press.
  6. Righetti, P. G., & Gelfi, C. (2001). 14. Electrophoresis. In Helmut Guenzler & A. Williams (Eds.), Handbook of Analytical Techniques (pp. 346–347). WILEY-VCH Verlag GmbH.
  7. Westermeier, R., Gronau, S., Becket, P., Buelles, J., Schickle, H., & Theßeling, G. (2005). Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations (4th, revised ed.). Wiley-VCH Verlag.
  8. Ven, S., & Rani, A. (2012). Discriminatory Power of Agarose Gel Electrophoresis in DNA Fragments Analysis. In Gel Electrophoresis – Principles and Basics. https://doi.org/10.5772/36891

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