Evaluating Starchy Electrophoretic Gel Alternatives to Agarose Industry Gold Standard

By Bryan D. Milstead

Abstract

Gel electrophoresis is a common laboratory technique for various applications regarding the separation and subsequent evaluation of biomacromolecules (ThermoFisher Scientific, n.d.). Additionally, agarose gel electrophoresis is widely considered to be the “most effective” way in which scientists can separate DNA fragments across variegated base pair quantities (namely, 100 bp to 25 kb) (Lee et al., 2012). From an economic standpoint, gel electrophoresis was a $1.2 billion industry in 2020 (Edvotek, 2022) and will only continue to undergo amplification due to its feasibility and usage in secondary-school settings (Ward’s World, n.d.) as well as collegiate research facilities (Phillips et al., 2017). Yet, scientists have consistently recognized a lack of accessibility to electrophoretic “gold standards” — namely, agarose and polyacrylamide (Shayah et al., 2024; Ume et al., 2022; Djankpa et al., 2021) in developing nations that lack the funding or geopolitical means to obtain these substances. 

Considering how pervasive gel electrophoresis is within academia and even forensic biology labs to investigate suspect DNA (US National Institute of Justice, 2023), it is reassuring to know that scientists are actively devising alternatives that prioritize cost-effectiveness and quality. For example, researchers from the Syrian Journal for Science and Innovation manufactured gels using animal gelatin, and wheat starch, among many other materials of similar biochemical complexity (Shayah et al., 2024). However, multiple sources emphasize the need for further research to be conducted, to more effectively understand the implications of alternative electrophoretic apparatuses (Shayah et al., 2024; Djankpa et al., 2021). Therefore, this research paper will review existing gel electrophoresis properties, bridge connections between emerging initiatives, and explore the implications of future gel decisions. 

Introduction

To elaborate on the aforementioned characteristics of gel electrophoresis, it utilizes a porous, quasi-sieve substance known as a “gel” (YourGenome, n.d.) which, regardless of the exact material, is known for possessing some level of viscosity. During this process, an electric current is applied across the gel matrix that forces charged molecules like DNA or RNA to migrate toward the oppositely charged electrode, usually in a negative-to-positive manner. However, charge isn’t the only factor that influences molecular movement — size, shape, buffer conductivity, and electroendosmosis can provide a multifaceted set of results (Lee et al., 2012). Once the gelling process concludes, the specific length of a biomacromolecule can be delineated via UV-light visualization with an appropriate dye (Lee et al., 2012). 

Figure 1: A comprehensive diagram of the gel electrophoretic process (Rogers, 2025). Notice how the movement travels from the negative electrode (indicated by the “-” sign) to the positive electrode (indicated by the “+” sign). The right-side portion of this figure reinforces the relationships between biomacromolecular length, weight, and travel distance, indicating that samples with greater values of the former two characteristics will result in an inversely proportional decrease in gel migration capabilities. This circumstance can be predominantly attributed to viscous drag, represented by a modified Stokes’ Law equation to describe electrophoretic mobility and hydrodynamic radii (University of Texas, n.d.). 

A specific medium, agarose, is the quintessential ingredient for casting gels (ThermoFisher Scientific, n.d.). It is an optimal choice due to having a large pore size and sufficient strength despite being submerged in saline buffer fluid (Block Scientific, n.d.). Researchers Patricia Barril and Silvia Nates highlight the non-toxicity and efficiency of agarose gels as “advantages”, with the high cost and fuzzy band size due to increased porosity as “disadvantages” (Barril et al., 2012). Furthermore, there is a risk of the gel melting, as agarose often succumbs to the high temperatures that an electric current administers throughout the buffer fluid (AAT Bioquest, 2024). This phenomenon can be exacerbated by human error in running the gel for too long and/or at an exceedingly high voltage (Douglas, n.d.).

It is also important to note that agarose is derived from the seaweed genera Gelidium and Gracilaria, which both belong to the red algae, or Rhodophyta, phylum (Science Direct, n.d.). The drawbacks with the former stem from its overall undesirable aquaculture traits: being small, slowly growing, and preferring rocky as well as shady areas which can be uncommon in the tropics of sub-Saharan Africa (Science Direct, n.d.). Contrariwise, the latter (Gracilaria) is prone to rapid growth rates and maintains a robust tolerance to temperature, light, and salinity, but requires labor-intensive frameworks of nets that have to be established underwater (Science Direct, n.d.). An overexploitation of seaweed biomass already exists in global agar production efforts (Sousa et al., 2021) but, ironically enough, is almost nowhere to be found in African countries (except Morocco and South Africa, two coastal nations). Most production occurs in East Asia or across the Americas (Hernández-Carmona et al., 2013), emphasizing the imperative that scientists must find feasible solutions to electrophoretic experiments, no matter their location. 

Exploring Alternatives to Agarose Gel: Variability, Chemistry, and Methodology

Researchers from the University of Cape Coast, in Cape Coast, Ghana, assessed the capabilities of corn starch as a substitute for agarose in DNA gel electrophoresis (Djankpa et al., 2022). In their study, they formulated a novel protocol to prepare industrial corn starch combined with a boric acid and sodium hydroxide buffer, in which the sediment retained after the mixing process was dried and ground into a smooth powder (Djankpa et al., 2022). Boiling-hot TAE (Tris-Acetate EDA), a textbook buffer in nucleic acid electrophoresis (Millipore Sigma, n.d.), was added to the aforementioned starch sediment and allowed to cool overnight, coagulating into a gelatinous slab. The results of their experiment were quite promising: the modified starch gel successfully formed wells (via comb placement) and allowed DNA samples to migrate through its saccharidic complex (Djankpa et al., 2022). However, the starch gel, in the researchers’ own words, appeared “cloudy” and “translucent” compared to the more “transparent” agarose gel, preventing a clear evaluation of DNA (Djankpa et al., 2022). 

 One might postulate that the structural properties of corn starch are the reason why the above comparison exists. Normal corn starch consists of roughly 75wt% amylopectin and 25wt% amylose (Sandhu et al., 2007), with the former being large and highly branched, whereas the latter is smaller and linear (Djankpa et al., 2022). However, section three of this research paper, “Connection Between Retrogradation Temperatures and Amylose-Amylopectin Ratios”, explains the nuances of factors that supersede the effects of amylopectin-amylose ratios. Overall, both polysaccharides swell when exposed to excess water, especially after undergoing a heating and cooling process — this phenomenon is known as retrogradation (Vanier et al., 2017). 

Starch retrogradation can result in gels with high levels of opacity and a thick consistency, depending on the speed as well as intensity of the recrystallization process (Vanier et al., 2017). Light is chaotically scattered throughout the disordered starch networks, increasing turbidity (Ulbrich et al., 2020). Conversely, agarose gel contains alternating 1,3-linked β-D-galactopyranose and 1,4-linked 3,6-anhydro-α-L-galactopyranose units, which has a uniform structure that allows for the more even passage of light (Sigma Aldrich, n.d.). In addition, amylopectin and amylose form relatively strong covalent bonds (BYJU’s, n.d.), a stark contrast from the weaker, intermolecular hydrogen (glycosidic) bonds that link β-D-galactopyranose and 3,6-anhydro-α-L-galactopyranose units together (Armisen et al., n.d.). Covalent bonds are in fixed positions and require orbital overlapping, indicating that the atoms are extraordinarily close to each other (FuseSchool – Global Education, 2016), which could explain the opacity of electrophoretic corn starch gel. In essence, denser substances possess a higher refractive index, bending or scattering light (Science Learning Hub, 2012). 

Figure 2: A comparison between the corn starch gel (a) and agarose gel (b), from the scientists at the University of Cape Coast, Cape Coast, Ghana (Djankpa et al., 2022). Notice the nebulous nature of the corn starch gel.

Researchers from Nigeria (Nigerian Defence Academy, Ahmadu Bello University) examine the feasibility of cassava (Manihot esculenta) and sweet potato (Ipomoea batatas) starch in the separation of DNA (Ume et al., 2022). In this study, cassava and sweet potato were selected due to being adequate sources of soft and transparent gels (Ume et al., 2022) and Nigeria’s role as a leading producer of these crops (National Root Crops Research Institute, 2023). The gelling temperature of two different cassava types (rubber and Opokopo) and sweet potato types (white and yellow) were determined by amalgamating them with lower quantities of agarose and agar-agar, as well as TBE (Tris-Borate-EDTA) buffer (Ume et al., 2022). Gels were subsequently formed using various novel protocols. While the yellow sweet potato gel boasted the largest pore sizes, rubber cassava and rubber cassava-agarose aggregate were clearer than any other starch samples, except agarose (Ume et al., 2022). Even so, rubber cassava starch was unable to form a durable gel without the addition of some amount of agarose (Ume et al., 2022).

Figure 3: The following figure is extracted from “Table 3” of the aforementioned results (Ume et al., 2022). Notice how the RCagar (rubber cassava and agar) mixture formed a clear, semi-solid gel in a relatively moderate gelling time, indicating its viability as an electrophoretic alternative.

Based on previous conclusions regarding starch content and amylase-amylopectin bonding, one seemingly abnormal data point in Figure 3 is the WSPagar at 4% starch concentration with the highest starch and lowest agar-agar value, yet it maintains a solid gel structure almost identical to that of agarose (Ume et al., 2022). It is important to note that the starch concentration of Djankpa et al.’s study was 12%, while Ume et al.’s study was 3.6%. Additionally, Ume et al.’s study blended agar-agar with their chosen starch, rather than replacing it altogether. Nevertheless, there are biochemical complexities that make cassava starch (with its roughly 82% amylopectin content (RIKEN, 2021) or 98% (AngelStarch, n.d.) depending on the source) clearer than corn starch. Cassava starch gelatinizes at a lower temperature than corn starch (AngelStarch, n.d.), rendering the retrogradation process less intense, and thereby mitigating opacity levels. 

At higher temperatures, starch granules absorb water and swell due to the disruption of hydrogen bonds within amylopectin chains (Jia, 2023). This allows for new hydrogen bonds to form between water and starch molecules (Xu et al., 2020), creating a disorderly combination of crystalline and amorphous structures. Higher amylose contents can become counteractive by leaching out of these starch granules into the surrounding material, reducing the ordered starchy structure even further (USAPulses, n.d.). These circumstances increase the turbidity (Chatterjee, 2024; Wang, 2015). 

Figure 4: A visual representation of the starch gelatinization process. From left to right: starch granules are normal (I) until swelling (IIa) followed by amylose leaching (IIb) and retrogradation (IIIa) and a predominantly amorphous structure with some crystallization (IIIb) (Orm, 2021). In the case of electrophoretic gel casting, IIIb would be almost completely amorphous due to the quick cooling time which prevents extensive recrystallization (Bento, n.d.).

Conclusion, and Implications for Future Gel Exploration:

The importance of starch as an alternative to agarose gel electrophoresis is crucial based on a wide range of locational, economic, and governmental factors. While numerous sources have made strides in establishing starch gel protocols for DNA experimentation, a thorough analysis of the thermochemistry and material science behind their negative, neutral, or positive results has not yet been conducted. This study aims to (1) highlight the intricacies of starch properties at different temperatures; (2) explore corn and cassava starches as aggregates with other substances; (3) emphasize the interconnectedness between retrogradation and amylase-amylopectin ratios; and (4) encourage the broader scientific community to pursue research endeavors that revolve around sustainable solutions. Amylose-amylopectin ratios and structures alone are not sufficient to determine the opacity of a starch gel and must be evaluated in the context of starch degradation.

  Future experimentation with starchy electrophoretic gels must be deeply attentive to gelatinization temperature(s) and its role in intensifying starch retrogradation. Furthermore, amylose-amylopectin ratios and the integration of agarose or agar-agar mediums should be considered. Lastly, if said integration is pursued, a process of “trial-and-error” is encouraged to create different percentages of agar-to-starch to optimize gelling capabilities; Ume et al. adopted this method in particular. This is because there exists very little literature about the protocols of hyper-niche starch variations (Ume et al., 2022). 

Edited by Lamisa Chowdhury

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