Five Considerations for the Nucleic Acid Gel Electrophoresis Process

The basic principles of electrophoresis imply that nucleic acid samples have different rates of mobility when they are of different sizes. However, nucleic acids with the same number of nucleotides but different sequence composition and conformation may have different mobilities during electrophoresis (Figure 1). 

• Sequence: AT-rich DNA may migrate more slowly than GC-rich DNA of the same size, especially in high-resolution electrophoresis. Similarly, DNA molecules with 4–6 adenosine repeats at approximately every 10 bp (called curved DNA) will migrate irregularly, especially in polyacrylamide gels [1,2]. Their anomalous migration is likely due to sequence composition affecting their molecular conformation. 
• Conformation: The migration of DNA molecules of the same sequence but differing conformations, such as circular and linearized plasmids, is affected by the compactness of each conformation as they move through the gel pores. Highly compact supercoiled molecules migrate the fastest, followed by flexible linear and open circular molecules (Figure 1). This differential migration may be exploited to examine the integrity of plasmid DNA after isolation, since intact plasmid DNA is desirable in applications like transfection of mammalian cells for gene overexpression.

The size and desired resolution of nucleic acid samples to be separated will often drive the selection between agarose or polyacrylamide gels. In general, higher gel percentages are more effective for resolving smaller molecules. Tables 1 and 2 summarize properties to consider when selecting agarose and acrylamide gel reagents for nucleic acid electrophoresis.

Gel thickness and well size may also influence electrophoresis results, with both agarose and polyacrylamide gels.

In general, thicker gels may cause bands to diffuse, due to more heat generated during gel runs. Suboptimal visualization may also occur due to a high background of gel stain or the length of time needed to stain and/or destain the gel (if post-electrophoresis staining is performed). For agarose gels, a thickness of 3–4 mm generally works well, and gels thicker than 5 mm are not recommended. The thickness of polyacrylamide gels is defined by spacers for gel casting plates supplied by the manufacturers, the most common of which are 0.75 mm, 1.0 mm, and 1.5 mm.

The size of the well, defined by the shape of the gel comb, affects not only how much sample can be loaded but also resolution of the bands. While larger wells accommodate increased sample loading, they may produce thick bands, reducing band resolution and creating smears. On the other hand, long and narrow wells accommodate smaller sample amounts but often provide sharper and more well-defined bands for better resolution. More compact samples also offer higher band intensity from less input.

An electrical field is applied for the electrophoretic separation of nucleic acid molecules. Hence, electrical parameters can impact sample migration and resolution of its constituent fragments [5,6].

The following equations, derived from Ohm’s Law, may be used to express the relationship between voltage (V), current (I), and power (P), all of which can influence results of electrophoresis.

1. Gel electrophoresis utilizes the principles of resistance and heat to separate DNA, RNA, or proteins based on size and charge. Resistance is affected by factors such as buffer conductivity, temperature, and gel properties.
2. Heat generation in gel electrophoresis is directly proportional to power consumption and is influenced by buffer conductivity, applied voltage, and resistance.

Gel electrophoresis is affected by constant voltage, power, and current on the system (Table 3). It's important to note that regardless of the electrical parameter set by the power supply, the voltage should be capped slightly below the maximum value that the system can handle to avoid overheating and damage to equipment and samples. A recommended practice is to set electrical running parameters high enough for efficient sample separation without generating excessive heat.

Table 3. Voltage, power, and current in gel electrophoresis

Fluorescent dyes are often used to visualize nucleic acid samples during or after electrophoresis. In addition to sensitivity, characteristics of the dyes such as excitation wavelength, binding affinity, and rate of gel penetration can impact workflow and applications of electrophoresis (Table 4) [7].

Table 4. Common properties of fluorescent dyes used in nucleic acid staining and the implications on gel electrophoresis process

Ethidium bromide (EtBr) is a fluorescent dye widely used in nucleic acid electrophoresis because of its short staining time (~30 min) and high sensitivity (detects ~1 ng of double-stranded DNA per band). 

Despite its advantages, EtBr has some drawbacks, and alternatives must be considered.

• Mutagenicity and disposal—EtBr is highly mutagenic, which makes it hazardous to handle. In addition, mutagenic reagents require special disposal measures. So fluorescent dyes such as the SYBR Safe DNA gel stain offer a safer workflow. 

• UV damage—Samples stained with EtBr must be viewed with UV light, which causes structural damage to nucleic acids. Other fluorescent dyes that can be excited with lower-energy blue light and detect with equal or higher sensitivity (Figure 4). As such, excitation with blue light in electrophoresis can help improve success in downstream applications such as cloning and sequencing.

Figure 4. Excitation and emission spectra of common nucleic acid stains. SYBR Safe and SYBR Gold stains can be excited maximally by blue light and to a lower extent by UV light.

In summary, selecting appropriate reagents, parameters, and methods, in addition to the workflow setup, is vital to achieving optimal results in electrophoresis for the separation and analysis of nucleic acids.

1. Carrera P, Azorín F (1994) Structural characterization of intrinsically curved AT-rich DNA sequences. Nucleic Acids Res 22(18):3671–3680.
2. Stellwagen NC (2009) Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution. Electrophoresis 30 Suppl 1:S188–195.
3. Lee SV, Bahaman AR (2012) Discriminatory Power of Agarose Gel Electrophoresis in DNA Fragments Analysis. In: Magdeldin S (editor), Gel Electrophoresis: Principles and Basics. Rijeka: InTech. pp 41–56.
4. Chory J, Pllard JD (1999) Resolution and Recovery of Small DNA Fragments. In: Ausubel FM et al. (editors) Current Protocols in Molecular Biology. Supplementary 45: Unit 2.7.1–2.7.8.
5. Thermo Fisher Scientific Inc. (2015) Protein Gel Electrophoresis Technical Handbook.
6. Sheehan D (2009) Electrophoresis. In: Physical Biochemistry: Principles and Applications. West Sussex: Wiley. pp 147–198.
7. Thermo Fisher Scientific Inc (2010) Nucleic Acid Detection and Analysis. In: Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies. pp 349–360.