by K Chico

Gel electrophoresis is a technique that uses electrical current to separate DNA, RNA or other proteins based on their size, shape, and charge. Gel construction is simple, quick, and relatively inexpensive and finished gels can have the consistency of a firm Jello to a rubberier substance, depending on concentration. Additionally, certain parameters of gels, like concentration and dimension, can easily be tuned depending on experimental need. Historically, starch gels containing potato starch have been used for zone electrophoresis, and more recently, research on corn starch as a gel medium has been conducted. Even so, agarose and polyacrylamide gels remain the gold standards.

 # Agarose Gel Agarose is a natural linear polysaccharide that can be isolated from some seaweeds. An agarose gel is prepared by pouring the warm liquid agarose solution into a casting tray with a comb that molds the wells for each sample. Upon casting, these sugars become interlinked to form pores, and the gel will set as it cools. Agarose gels typically utilize a horizontal apparatus where dimensions can be easily modified. A larger gel, for example, allows samples to run on the gel for longer durations without running off into the buffer. The horizontal setup additionally makes this technique suitable for immune electrophoresis. Recently, a vertical system combined with sodium dodecyl sulfate (SDS) has been developed to separate very large proteins (200-4,000 kDa).

Although agarose gels are non-toxic and easy to handle, they do not exhibit great uniformity in pore size, which becomes smaller with increasing agarose concentration. Likewise, agarose gels can only separate double-stranded (ds) DNA; though they maintain their status as matrix of choice for the separation of nucleic acids since polyacrylamide gels can be toxic in the non-polymerized form and complex to prepare. Agarose gels have a lower resolving power than polyacrylamide gels but have a greater range of separation. Agarose gels are generally suitable for separating molecules from roughly 50 bp-20 kbp, which makes them increasingly useful for large DNA fragments, such as the products of PCR. In some techniques like pulsed field gel electrophoresis, resolution of over 6 Mb is also possible using agarose gels.  

Polyacrylamide gels are created by the polymerization of acrylamide monomers in the presence of bisacrylamide, a crosslinking agent. Polyacrylamide gels are created by either pouring the warm polyacrylamide solution in the space between two parallel vertical glass plates, or by using a glass cassette. The chemical crosslinking of acrylamide and bisacrylamide creates a sieved membrane in which fragments become trapped. Polyacrylamide gels typically utilize a vertical apparatus, where a comb is placed on top of the gel to create each sample well, and samples run downward instead of horizontally.

The pore size of polyacrylamide gels is incredibly consistent and reproducible and is directly related to the ratio of acrylamide to bisacrylamide (which inherently determines the concentration of the gel). Though construction of these gels is more expensive in time and resources, they often provide more reproducible results over agarose counterparts. Polyacrylamide gels can also separate DNA in ds and single strand (ss) form and can be further modified to acquire superior resolution for nucleic acids. For example, denaturing polyacrylamide gel can distinguish ssDNA molecules that are only one nucleotide different, allowing for subsequent Sanger sequencing.

Polyacrylamide gels are widely used for small nucleic acids (e.g., miRNA and tRNA) as well as other proteins because they are more adaptable to accommodate several aspects of protein characterization. In native polyacrylamide gel electrophoresis (PAGE), proteins can be distinguished based on the natural or denatured confirmation using non-denatured gels. Alternatively, proteins can also be separated based on their size instead, when using higher percentage (10%-20%) gels with SDS, a denaturing agent.  

 # References Manual for Starch Gel Electrophoresis: A Method for the Detection of Genetic Variation
Assessment of corn starch as substitute for agarose in DNA gel electrophoresis
Gel Electrophoresis
Principles and Techniques of Biochemistry and Molecular Biology Seventh edition