Nucleic Acid Electrophoresis Additional Considerations–7 Aspects
The nucleic acid electrophoresis workflow requires the execution of several steps, each of which may affect the result of nucleic acid separation. This page discusses 7 additional considerations related to samples, reagents, and operating parameters, including:
Nucleic acid sequence and conformation
Gel reagent properties
Gel thickness and pore size
Type of running buffer
Voltage, current and power in gel operation
Nucleic acid staining property
Gel staining method
1. Sequence and conformation of nucleic acid samples
The basic principle of electrophoresis shows that different sizes of nucleic acid samples have different mobility. However, nucleic acids with the same number of nucleotides but different sequence compositions and conformations may also have different mobility during electrophoresis (FIG. 1).
Sequence AT-rich DNA may migrate more slowly than GC-rich DNA of the same size in high-resolution electrophoresis. In addition, DNA molecules with 4-6 adenosine repeats approximately every 10 bp (known as DNA coilings) will exhibit irregular migration, especially in polyacrylamide gels 1,2. The anomalous migration may be caused by the sequence composition affecting the molecular conformation.
Conformation: The migration of DNA molecules with the same sequence but different conformations, such as ring and line plasmids, is affected by the closeness of each conformation when passing through the gel pore. Highly dense superhelical molecules migrate the fastest, followed by flexible linear and open-loop molecules (FIG. 1). This difference in migration rate can be used to test the integrity of plasmid DNA after isolation, since intact plasmid DNA is very useful in applications such as transfected gene overexpression in mammalian cells.
Figure 1. Electrophoretic migration of identical DNA with different conformations. (A) Electrophoresis of circular, linear and supercoiled plasmid DNA with incisions. (B) Conformation of relaxed circular, linear and supercoiled plasmid DNA. The incised plasmid is a relaxed and open ring-shaped conformation with large volume and slow migration in the gel. The migration speed of linear particles is slightly faster. Intact plasmids are the most compact and migrate the fastest.
2.The properties of agarose and acrylamide reagents can be used as electrophoretic related properties for electrophoresis
The choice of agarose or polyacrylamide gel depends primarily on the size of the nucleic acid sample to be separated and the resolution desired. In general, high percentage gels are suitable for the separation of small molecules with good results. Table 1 summarizes the properties to be considered when selecting agarose and acrylamide gel reagents for nucleic acid electrophoresis.
Table 1. Properties of agarose and acrylamide gel reagents for electrophoresis 3,4.
Agarose | |
Property | Implications |
Clarity | Agarose forms a translucent gel. Therefore, agarose with a higher definition specification ensures minimal fluorescence background during gel visualization and documentation. |
Electroendosmosis（EEO） | During electrophoresis, the interaction between buffer ions and charge molecules on the surface and inside the agarose matrix affects the movement of buffer toward the electrode, a process known as electroosmosis or EEO. Since the positively charged ions (cations) of the buffer flow in the opposite direction to the nucleic acid (Figure 2A), the agarose with a higher negative charge will affect the movement of cations, thus affecting the separation of large nucleic acid molecules (> 10 KB). The EEO value can be regarded as an indirect representation of the number of negatively charged groups in agarose. The oxygen atoms on the side chains of agarose (represented by X in figure 2B) can carry hydrogen (X = H), or negatively charged groups such as sulfate (SO3 --) and pyruvate (CH3COCOO --). Since negative charges attract buffer cations, the presence of these groups on agarose reduces the efficiency and resolution of nucleic acid separation. |
Gel point | The gel point refers to the temperature at which the agarose solution forms a gel. The higher the gel ratio, the higher the gel point. |
Gel strength | Gel strength is expressed in units of force (g/cm2), which refers to its ability to resist crushing, depending on the agarose concentration. The stronger the gel, the easier it is to work with. |
Genetic quality | Genetic quality (GQ) indicates that whether agarose is applicable to molecular biology is mainly based on the level of pollutants and enzyme inhibitors. |
Melting point | Melting point refers to the melting temperature of agarose. Since agarose gels melt when heated, the melting point is always higher than the gel point. Low melting point agarose (LMP) is a special agarose whose melting temperature (~ 25°C) is significantly lower than that of other standard agarose. LMP agarose also has a low gel point, which is very suitable for large nucleic acid extraction and ligation and other intra-gel enzyme reactions. |
Acrylamide gel reagent | |
Property | Implications |
Molecular biology grade | Use high-quality, molecular biograde reagents that have been tested for nuclease activity and the presence of contaminants. It can protect the integrity of nucleic acid samples during electrophoresis. |
Stability/shelf life | It is commercially common to inject some kind of gas into polyacrylamide stock solution to prolong its stability. If you make your own polyacrylamide stock solution in the laboratory, be careful to use it within a few months, otherwise it will break down into acrylic acid over time. Acrylamide and bisacrylamide powder or solution should be stored in a container that is protected from light.
Ammonium sulfate (APS) solution is best used in preparation to form free radicals to initiate gel polymerization. The prepared stock solution can be stored for about one month at 4°C, but the efficiency decreases with time.
Tetramethylethylenediamine (TEMED) is a free radical reagent that stabilizes gel polymerization and should be stored sealed to prevent oxidation. |
Total percentage of monomers, w/v (%T) | The total content (% T) of acrylamide monomer and cross-linked bisacrylamide in solution determines the pore size of the polyacrylamide gel. For example, a 10% polyacrylamide gel consists of 10% (w/v) acrylamide and bisacrylamide. The higher the %T, the smaller the pores, and the higher the resolution for separating small molecules (learn more: polyacrylamide gels recommended %). |
Percentage of crosslinker(%C) | %C is the ratio of crosslinking dose to total monomer (w/w). In specified %T, the higher %C is, the smaller the pores are. %C also refers to the ratio of acrylamide to bisacrylamide (e.g., 5 %C is 19:1). Polyacrylamide gels at 5% C(19:1) and 3.3% C(29:1) are commonly used for nucleic acid electrophoresis. |
FIG. 2. (A) Movement of buffer cations relative to nucleic acids during electrophoresis. (B) In the position of oxygen in the structure unit of agarose, negatively charged groups can be carried (indicated by X).
Gel thickness and pore size, including agarose and polyacrylamide gels, can also affect electrophoretic results 3.
Generally speaking, thicker gels generate more heat during operation, which can lead to band diffusion. Poor visualization may occur due to the high background of gel staining or the time required for gel staining and/or decolorization, such as post-electrophoretic staining. For agarose gels, 3-4 mm thickness is preferred; gels larger than 5 mm are not recommended. The thickness of the polyacrylamide gel is determined by the spacer provided by the manufacturer for gel filling, most commonly 0.75 mm,1.0 mm, and 1.5 mm.
The pore size determined by the comb shape not only affects the loading capacity of the sample, but also affects the resolution of the strip. Although larger holes can accommodate larger sample loads, they also produce coarser strips, reducing strip resolution and creating smudges. While long and narrow holes can accommodate a small sample volume, they can provide a clearer band for better resolution. Reducing the amount of high-density sample injected provides a higher intensity band.
4.Type of running buffer
Two running buffers commonly used in nucleic acid electrophoresis are Tris-EDTA acetate (TAE) and Tris-borate EDTA (TBE)5 (see the gel running steps in the previous section). Low molecular weight samples (e.g. DNA＜1000bp) TBE buffer with higher ionic strength and buffering capacity had better separation effect; Large DNA fragments were not well separated in TBE buffer. Denaturing electrophoresis for the separation of molecules that give rise to secondary structures, such as RNA, usually uses TBE buffer, since polyacrylamide gels primarily use TBE buffer supplemented with 7-8 M urea or similar denaturing agents to maintain the single-stranded state of nucleic acids.
For nucleic acids with large molecular weights (e.g., DNA≥12-15 KB), low field strength (1-2 V/cm)TAE buffers are preferred. TAE buffer can promote gels to produce larger size pores, reduce electroosmosis (which is a relatively less charged buffer), and reduce the strength of the electric field to reduce the tendency of macromolecules to produce stains 6.
5.Electrical operation parameters: voltage, current, power
The sample is separated by electrophoresis, and the negatively charged nucleic acid migrates to the positive electrode after applying an electric field. Thus, the electrical parameters of electrophoresis can influence the migration of the sample and the resolution of the fragments that compose it 7,8.
The following equations, derived from Ohm's law, can be used to show how voltage (V), current (I), and power (P) affect the electrophoretic results.
Voltage = current X resistance, i.e. V = I x R
Power = current x voltage, namely P = I x V
Since V = I x R, power can also be expressed as P = I2 x R.
During gel operation, resistance (R) is a fixed parameter of the system. For example, the buffer of the system (conductivity and buffer capacity), temperature, gel properties (percentage, height, length, quantity, cross section), etc., all affect resistance. In a conductive medium, higher temperatures allow more current to flow, so the resistance decreases as the temperature increases. However, the resistance may vary during gel running.
Another important factor affecting electrophoresis is heat. The heat generated is proportional to the energy consumed by the system and depends on the buffer conductivity, applied voltage and resistance. The higher the conductivity of the buffer (especially if it consists of small ions), the higher the current. High voltage and low resistance can also improve current. The increase in overall current increases the amount of energy and heat produced by the system.
Table 2 describes how resistance and heat affect the effects of constant voltage, power supply, and current on the electrophoresis system. Whether or not the power supply provides constant electrical parameters, the voltage ceiling should be slightly lower than the maximum of the system to avoid overheating leading to damage to equipment and samples. It is generally recommended to set electrical parameters that allow efficient sample separation without excessive heat generation.
Table 2. Voltage, power and current of electrophoresis.
Voltage (V) | Power (P) | Current (I) | |
Description | V = I x R Corresponds to the electrical potential difference between the two electrodes of a gel system | P = I x V or P = I2 x R Measures the rate of energy conversion, which is correlated to heat generated by the system | I = V/R Denotes the flow of buffer ions and is directly correlated to the applied voltage |
Electrophoresis implications | Voltage contributes to the field strength (V/cm). Higher voltage moves charged molecules faster. Constant voltage is recommended, as it offers the most control over the speed of sample migration. Variation in the resistance (R) (e.g., from different numbers and cross-sections of gels) in a given system is compensated for by changes in the current (I) at the constant voltage (V = I x R), keeping the rate of sample migration relatively constant. | Constant power prevents overheating of the system but may result in variable sample mobility. Depletion of buffer ions (decreased current) over a lengthy gel run may result in a progressive increase in voltage to maintain constant power (P = I x V). Isoelectric focusing (IEF) of proteins represents an application of constant power, where a gradual increase in voltage is desired to “focus” samples into narrow zones at the completion of gel runs. | Current contributes to power (P = I2 x R), by the order of magnitude of two. Constant current keeps power consumption and heat generation of the system relatively constant (in continuous nongradient gels for nucleic acids). In discontinuous or gradient gel electrophoresis for proteins, constant current may be useful to stack samples. Since I = V/R, when samples enter higher-percentage gels (having increased resistance), the voltage also increases to keep the current constant, exerting a larger electrical force on the samples in the process. |
6.Common properties of nucleic acid fluorescent dyes
In electrophoresis, fluorescent dyes are often used to stain nucleic acid samples to visualize them. In addition to sensitivity, the characteristics of dye, such as excitation wavelength, binding affinity and gel penetration speed, can also affect the workflow and application of electrophoresis (Table 3)9.
Table 3. Common characteristics of fluorescent dyes used for nucleic acid staining.
Property | Implications |
Binding affinity | The binding affinity of the dye is very important because the fluorescence of the dye is enhanced when it binds to the sample for easy observation. In general, nucleic acid dyes have a higher affinity for double-stranded molecules, such as DNA, than for single-stranded molecules, such as RNA, because they bind more easily to double-stranded helical molecules. For RNA electrophoresis, the specificity and sensitivity of RNA detection can be improved by unique dyes with high affinity for single-stranded molecules. |
Compatibility with denaturants | Urea and formamide are commonly used as denaturants for RNA electrophoresis. In denaturing electrophoresis, the anti-quenching effect of denaturing agents on dyes should be considered to improve the effectiveness. Otherwise, the denaturant should be removed by washing before staining. |
Dynamic range | The dynamic range indicates the order of magnitude at which linear detection of sample volume occurs. Thus, dyes with a wider dynamic range allow more accurate quantification of bands in the gel. |
Excitation wavelength | Longer wavelengths produce lower energy and thus less damage to nucleic acids. Therefore, compared with UV excitation, the use of blue light excitation dye can protect the integrity of the sample. And will have a significant impact on downstream applications, such as cloning efficiency. |
Gel penetration | In cases where post-electrophoretic staining is used, dyes that quickly penetrate the gel not only shorten the workflow, but also stain thicker and higher percentage gels better. |
Intrinsic flurescence | Low endogenous fluorescent dyes have low background in gel staining, which can improve detection and avoid decolorization. |
Mutagenicity | Fluorescent dyes used in nucleic acid staining are usually mutagenic by their interactions. Dyes with lower mutagenic and non-hazardous properties are safer and easier to handle. |
7.Binding of dye to sample: Methods for in-gel and post-electrophoresis staining
Two common methods of staining nucleic acid samples include:
(1) Within the gel, the stain is combined with the gel (and running buffer)
(2) After electrophoresis, the gel was soaked and stained separately after running.
Both methods have advantages and disadvantages, as shown in Table 4
Methods | Benefits | Considerations |
In-gel staining | More convenient Faster workflow Requires less stain | Stain may run off after a long run Stain can be used only once May alter sample mobility |
Post-electrophoresis staining | Provides more accurate analyses of molecular sizes Allows reuse of staining solutions or use with multiple gels | Longer workflow Requires more stain May amount to more hazardous waste |
In-gel staining is more convenient and requires less dye for visualization. However, positively charged stains migrate in the opposite direction to nucleic acids and can affect the detection of low molecular weight samples, especially when run for long periods (FIG. 3A). In addition, dyes bound to nucleic acids can alter the migration of the sample, known as gel migration, resulting in a sample size that is not realistic (FIG. 3B). In addition, the addition of a high level of embedded dye to the gel may alter the conformation of supercoiled plasmid DNA and alter its mobility (apparent molecular weight) in electrophoresis10
Since post-electrophoresis staining does not affect the detection of samples, it is the preferred method for accurate determination of sample size.
However, the process takes longer to work, requires more dyes, and produces more hazardous waste when dyes such as ethidium bromide are used.
Figure 3. Results of nucleic acid gel electrophoresis with embedded dye. (A) Ethidium bromide added to the gel moves in the opposite direction to the nucleic acid. The yellow line marks the boundary between the ethidium bromide fluorescence background and the gel region where the dye has been depleted. (B) A large embedded dye, when added to the gel rather than applied to post-electrophoretic staining, affects the migration of the sample band. To illustrate this effect, two molecular weight standards were run side by side in two gels. The high molecular weight molecular weight standard (1 column) had similar migration in both gels (the blue line indicates the reference band, indicating the band location). During electrophoresis, low molecular weight molecular weight criteria (2 columns) migrate differently in the presence of dye.
In conclusion, in addition to workflow Settings, the selection of appropriate reagents, parameters and methods in electrophoresis is essential for optimal nucleic acid separation and analysis.
References
7.Thermo Fisher Scientific Inc. (2015) Protein Gel Electrophoresis Technical Handbook.
8.Sheehan D (2009) Electrophoresis. In: Physical Biochemistry: Principles and Applications. West Sussex: Wiley. pp 147–198.
9.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.