In the nucleic acid electrophoresis workflow, several steps are performed sequentially, each of which could impact the outcome of nucleic acid separation. This article addresses five considerations associated with samples, reagents, and running parameters.
Effects of conformation: Supercoiled vs circular vs linear DNA in gel electrophoresis
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.
Figure 1. Electrophoretic migration of the same DNA in various conformations. (A) Electrophoresis of nicked circular, linear, and supercoiled plasmid DNA. (B) Conformation of relaxed circular, linear, and supercoiled plasmid DNA. Nicked plasmids assume a relaxed, open circular conformation and take up the most volume, migrating most slowly through the gel; linearized plasmids move through the gel at a slightly higher rate; intact, supercoiled plasmids, being the most compact, migrate the fastest.
Effects of gel type: Properties of acrylamide and agarose used in electrophoresis
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.
Table 1. Properties of agarose gel reagents that influence electrophoresis [3,4]
| Property | Implications |
|---|---|
| Clarity | Agarose forms translucent gels. Therefore, agarose with a higher clarity specification helps ensure minimal fluorescence background during visualization and documentation of the gel. |
| Electroendosmosis (EEO) | During gel electrophoresis, the movement of buffer towards an electrode can be affected by the interaction between buffer ions and charge molecules within and on the surface of the agarose matrix, known as electroendosmosis or EEO. Figure 2A illustrates how positively charged ions (cations) of the buffer flow in the opposite direction of nucleic acids. Agarose with higher negative charges can impact the movement of cations and the separation of large nucleic acids (>10 kb). The EEO value can be used as an indicator of the amount of negatively charged groups in the agarose. Figure 2B shows how oxygen atoms on the side chains of agarose may carry negatively charged groups such as sulfate and pyruvate (indicated by X). These negatively charged groups attract buffer cations, decreasing the efficiency and resolution of nucleic acid separation.
|
| Gel point | Gel point indicates the temperature at which an agarose solution forms a gel. The higher the gel percentage, the higher the gel point. |
| Gel strength | Gel strength, expressed in the unit of force (g/cm2), corresponds to the ability of a gel to withstand breakage and is dependent upon agarose concentration. The higher the gel strength, the easier it is to handle. |
| Genetic quality | Genetic quality (GQ) indicates whether agarose is suitable for molecular biology applications, based on levels of contaminants and enzyme inhibitors. |
| Melting point | Melting point is the temperature at which agarose melts. Since gelled agarose melts when heat is applied, the melting point is always higher than the gel point. Low melting point (LMP) agarose is a specific type of agarose that melts at a significantly lower temperature (~25°C) than standard agarose. LMP agarose also exhibits a lower gel point, which is helpful for extraction of large nucleic acids and setting up in-gel enzymatic reactions like ligation. |
Table 2. Properties of acrylamide gel reagents that influence electrophoresis [3,4]
| Property | Implications |
|---|---|
| Molecular biology grade | High-quality, molecular biology-grade reagents have been tested for nuclease activity and the presence of contaminants. This qualification can help protect the integrity of nucleic acid samples during electrophoresis. |
| Stability/shelf life | Commercially prepared stock solutions of polyacrylamide are often stabilized by infusion with a gas to prolong their stability. If you prepare polyacrylamide stock solutions in lab, they should be used within a few months because they break down to acrylic acid over time. Acrylamide and bisacrylamide, in powder or solution, should be stored in dark containers to protect them from light. The ammonium per sulfate (APS) solution is best prepared fresh, for free radical formation to initiate gel polymerization. The prepared solution may be stored at 4°C for about one month, but its efficiency decreases over time. Tetramethylethylenediamine (TEMED), a reagent that stabilizes the free radicals formed in gel polymerization, should be stored tightly capped to prevent oxidation. |
| Total percentage of monomers, w/v (%T) | The total percentage of monomeric acrylamide and crosslinking bisacrylamide in solution (%T) determines the pore size of a polyacrylamide gel. For example, a 10% polyacrylamide gel is composed of 10% (w/v) of acrylamide and bisacrylamide. The higher the %T, the smaller the pore size and higher the resolving power to separate smaller molecules. |
| Percentage of crosslinker (%C) | The %C refers to the amount of crosslinkers with respect to the total amount of monomers (w/w). At a given %T, the higher the %C, the smaller the pore sizes. %C may also be presented as the ratio of acrylamide to bisacrylamide (e.g., 5 %C as 19:1). Polyacrylamide gels of 5 %C (19:1) and 3.3 %C (29:1) are commonly used in nucleic acid electrophoresis. |
Table 1. Properties of agarose gel reagents that influence electrophoresis [3,4]
| Property | Implications |
|---|---|
| Clarity | Agarose forms translucent gels. Therefore, agarose with a higher clarity specification helps ensure minimal fluorescence background during visualization and documentation of the gel. |
| Electroendosmosis (EEO) | During gel electrophoresis, the movement of buffer towards an electrode can be affected by the interaction between buffer ions and charge molecules within and on the surface of the agarose matrix, known as electroendosmosis or EEO. Figure 2A illustrates how positively charged ions (cations) of the buffer flow in the opposite direction of nucleic acids. Agarose with higher negative charges can impact the movement of cations and the separation of large nucleic acids (>10 kb). The EEO value can be used as an indicator of the amount of negatively charged groups in the agarose. Figure 2B shows how oxygen atoms on the side chains of agarose may carry negatively charged groups such as sulfate and pyruvate (indicated by X). These negatively charged groups attract buffer cations, decreasing the efficiency and resolution of nucleic acid separation.
|
| Gel point | Gel point indicates the temperature at which an agarose solution forms a gel. The higher the gel percentage, the higher the gel point. |
| Gel strength | Gel strength, expressed in the unit of force (g/cm2), corresponds to the ability of a gel to withstand breakage and is dependent upon agarose concentration. The higher the gel strength, the easier it is to handle. |
| Genetic quality | Genetic quality (GQ) indicates whether agarose is suitable for molecular biology applications, based on levels of contaminants and enzyme inhibitors. |
| Melting point | Melting point is the temperature at which agarose melts. Since gelled agarose melts when heat is applied, the melting point is always higher than the gel point. Low melting point (LMP) agarose is a specific type of agarose that melts at a significantly lower temperature (~25°C) than standard agarose. LMP agarose also exhibits a lower gel point, which is helpful for extraction of large nucleic acids and setting up in-gel enzymatic reactions like ligation. |
Table 2. Properties of acrylamide gel reagents that influence electrophoresis [3,4]
| Property | Implications |
|---|---|
| Molecular biology grade | High-quality, molecular biology-grade reagents have been tested for nuclease activity and the presence of contaminants. This qualification can help protect the integrity of nucleic acid samples during electrophoresis. |
| Stability/shelf life | Commercially prepared stock solutions of polyacrylamide are often stabilized by infusion with a gas to prolong their stability. If you prepare polyacrylamide stock solutions in lab, they should be used within a few months because they break down to acrylic acid over time. Acrylamide and bisacrylamide, in powder or solution, should be stored in dark containers to protect them from light. The ammonium per sulfate (APS) solution is best prepared fresh, for free radical formation to initiate gel polymerization. The prepared solution may be stored at 4°C for about one month, but its efficiency decreases over time. Tetramethylethylenediamine (TEMED), a reagent that stabilizes the free radicals formed in gel polymerization, should be stored tightly capped to prevent oxidation. |
| Total percentage of monomers, w/v (%T) | The total percentage of monomeric acrylamide and crosslinking bisacrylamide in solution (%T) determines the pore size of a polyacrylamide gel. For example, a 10% polyacrylamide gel is composed of 10% (w/v) of acrylamide and bisacrylamide. The higher the %T, the smaller the pore size and higher the resolving power to separate smaller molecules. |
| Percentage of crosslinker (%C) | The %C refers to the amount of crosslinkers with respect to the total amount of monomers (w/w). At a given %T, the higher the %C, the smaller the pore sizes. %C may also be presented as the ratio of acrylamide to bisacrylamide (e.g., 5 %C as 19:1). Polyacrylamide gels of 5 %C (19:1) and 3.3 %C (29:1) are commonly used in nucleic acid electrophoresis. |
Effects of gel thickness and well size on the gel electrophoresis process
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.
Effects of power, current, and voltage in gel electrophoresis
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.
Voltage = current x resistance, or V = I x R
Power = current x voltage, or P = I x V
Power can be expressed as P = I2 x R, since V = I x R.
- 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.
- 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
| Voltage (V) | Power (P) | Current (I) | |
|---|---|---|---|
| Description |
|
|
|
| Electrophoresis implications |
|
|
|
Effects of fluorescent dyes on nucleic acid 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
| Property | Implications |
|---|---|
| Binding affinity | The binding affinity of a dye is an important factor because fluorescent enhancement is often observed upon dye binding to the samples. In general, nucleic acid dyes have higher affinity for double-stranded molecules (e.g., DNA) than single-stranded molecules (e.g., RNA), since it is easier to bind to double-stranded helices. For RNA electrophoresis, unique dyes with higher affinity for single-stranded molecules can increase specificity and sensitivity in RNA detection. |
| Compatibility with denaturants | Urea and formamide are typically used as denaturants in RNA electrophoresis. Dyes that are resistant to quenching by these denaturants should be considered in denaturing electrophoresis, for improved effectiveness. Otherwise, denaturing gels should be washed to remove denaturants prior to staining. |
| Dynamic range | The dynamic range represents the orders of magnitude in which linear detection of sample amounts occur. Therefore, dyes with a broader dynamic range allow more accurate quantitation of bands in the gel. |
| Excitation wavelength | Longer wavelengths exert lower energy, meaning less damage to nucleic acids. Thus, dyes excited using blue light protect sample integrity better than those excited by UV light. Damage to nucleic acids have pronounced implications in downstream applications, such as cloning efficiency. |
| Gel penetration | Dyes such as SYBR Gold Nucleic Acid Gel Stain that penetrates gels faster shorten the workflow and stain thick and high-percentage gels better when used in post-electrophoresis. |
| Intrinsic fluorescence | Dyes with low intrinsic fluorescence result in lower background in gel staining, circumventing the need to destain while improving detection. |
| Mutagenicity | Fluorescent dyes used in nucleic acid staining are often mutagenic due to their intercalating property. Dyes such as SYBR Safe DNA Gel Stain that have proven to be less mutagenic and classified as nonhazardous should be considered for added safety and convenient disposal. |
Fluorescent stains selection
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.
- Sensitivity—Fluorescent dyes that are higher in sensitivity than EtBr are usually better suited for detection of low amounts of samples (Figure 3). Single-stranded nucleic acids often require more samples and/or an intercalating dye for visualization because there is less base stacking. Therefore, dyes that preferentially bind to single-stranded nucleic acids can be good alternatives for sample detection in DNA gel electrophoresis.
Figure 3. Alternatives to ethidium bromide. Visual comparison of how much dsDNA is needed for detection using the following dyes: EtBr, SYBR Safe Stain, SYBR Green I Stain, and SYBR Gold Stain.
- 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.
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For Research Use Only. Not for use in diagnostic procedures.