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Over fifty years ago, DNA electrophoresis emerged as a molecular assay technique, established on fundamental electrochemical principles. The technique evolved from protein and RNA separation methods developed in the 1950s and 1960s, with Coleman and Miller pioneering the migration of DNA using a borax (sodium borate) solution. Subsequently, RNA and DNA electrophoresis adapted from protein electrophoresis methods, with Danna and Nathans employing these techniques in the early 1970s to analyze the structure of SV40 DNA fragments produced by new restriction endonucleases from Hemophilus influenza.

During electrophoresis, DNA is forced to pass through an agarose matrix due to exposure to an electric current. The negatively charged sugar-phosphate backbone propels DNA molecules toward the anode, predominantly relying on size for separation. The bigger the size of a DNA fragment, the slower it migrates. The application of higher voltage thereby accelerates the migration of samples. The difference in rate of migration enables efficient separation of DNA fragment length mixtures by electrophoresis. 

Slab gels are the most common form of DNA electrophoresis which involves molding agarose medium with conductive medium (buffer) and applying a voltage so that samples can migrate in parallel. During electrophoresis, DNA molecules are exposed to an electric field and migrate toward the anode due to its negatively charged phosphate backbone. Agarose gel electrophoresis needs several electrolytes to control pH and conductance. They are Tris base (2-amino-2-(hydroxymethyl)-1,3-propanediol), borate, acetate, chloride, glycine, sulfate and/or phosphate. The most commonly used conductive mediums for electrophoresis are Tris-borate-acid disodium EDTA (TBE) and Tris-acetate-acid disodium EDTA (TAE).

Tris base-based buffers, such as TAE and TBE, have been the dominant choice for electrophoresis throughout its history. However, these buffers have their own disadvantageous. TAE possesses lower buffering capacity which may extend electrophoretic times while in TBE generation of excessive heat is a primary problem. Heat not only creates a “runaway” positive feedback loop of current but also leads to sample diffusion and denaturation. It also causes poor gel integrity and limits the ability to run gels at high voltage. In an attempt to solve this problem, lithium borate buffer is proposed to replace TAE and TBE buffer.

Due to the low conductivity of lithium ions, it permits lithium acetate borate (LAB) to be used at high voltage and high speed for DNA separation so the time for separation is shortened. This is clearly shown in Figure 1 where DNA sample fragments (S1 – S4) migrate farther and the DNA fragments in the DNA marker (M) separate more distinctly in LAB buffer than in that of TBE buffer. This evidently shows that electrophoresis can be done more quickly and efficiently. Moreover, LAB buffer is much cheaper than Tris (2-amino-2-(hydroxymethyl)-1,3-propanediol) which is widely used in TBE buffer which costs almost RM1000 per kg. By using acetate and boric acid, the cost of preparation of buffer can be significantly reduced. 

TBE buffer is known to cause faster temperature elevation over time. The temperature of buffers was measured before and after 20 minutes of electrophoresis at 200 V and the rise of temperature was recorded. It was found that LAB buffer temperature increased more slowly and gradually while the TBE buffer showed a higher temperature rise compared to LAB and more drastically especially at 300 V (Figure 2). The presence of sodium ions from sodium EDTA in TBE buffer leads to the generation of excess current, thereby restricting the capability to run gels at high voltage. The consequential heat production imposes constraints on gel electrophoresis by limiting the voltage that can be applied to the gel system. Moreover, this heat generation has the potential to denature the sample and compromise the integrity of the gel.

LAB buffer is devoid of sodium EDTA and substitutes alkali metal cations for Tris which is lithium. Lithium is preferred over other alkali metal cations due to its large shell of hydration and low electrokinetic capacity, which provides lower conductance, improved tolerance for higher voltage, lowers running temperature, and enhanced electrophoretic separation efficiency. Compared to TBE, the alkali-metal ion mixture lowers the final buffer temperature, reduces the conductivity and decreases the time for electrophoretic separation.

In conclusion, it was evident that LAB buffer possesses low conductivity that permits its high voltage usage. This eventually will save time, energy and cost. LAB buffer could be the potential new generation DNA electrophoresis conductive medium in the future.