Nucleic acid electrophoresis is utilized in many research applications of molecular biology to examine experimental outcomes and, in some cases, to isolate and purify samples, before proceeding to a subsequent step. Therefore, applications of routine nucleic acid electrophoresis can be generally categorized as analytical or preparative, respectively, both of which rely on the technique to separate, resolve, and quantitate. These applications include:

1. Analytical applications to determine experimental outcomes

The analytical applications of nucleic acid electrophoresis provide ways to examine experimental results of a prior step before continuing the workflow or another set of experiments. This approach primarily relies on evaluating the presence or absence of desired bands in gels, their intensities, migration patterns, mobilities, and hybridization, as described below.

a. Determination of success in enzymatic synthesis, digestion, and cloning experiments

Electrophoretic analysis of nucleic acids is commonly carried out immediately after the following techniques (Figure 1) to determine experimental success and efficiency:

Figure 1. Common molecular biology applications in which electrophoresis may be used to determine experimental success.

Figure 2. Ligation efficiency determined by gel electrophoresis. Lambda DNA was first cleaved with HindIII, a type II site-specific restriction enzyme (lane 1). The sample was religated and analyzed by gel electrophoresis (lane 2); the fully ligated DNA is the most prominent band.

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b. Quantitation of nucleic acid samples by gel electrophoresis

Electrophoresis may be used to quantitate DNA or RNA bands of interest by utilizing a standard or ladder containing a known quantity of each fragment. The widely used spectrophotometric quantitation method can be skewed by the presence of contaminants such as nucleotides, primers, and other species. Electrophoresis separates the sample of interest from these contaminants, offering an alternative reliable quantitation strategy.

Gel quantitation is achieved by comparing the intensity of a band in the sample to a band in the ladder that is similar in size, to estimate the quantity of the sample (Figure 3A). Ideally, when using a ladder designed for quantitation, varying amounts of the ladder should be loaded to plot a standard curve of amount vs. intensity, for more precise quantitation (Figure 3B). In addition, staining nucleic acids with a fluorescent dye that has high sensitivity and a wide dynamic range can further improve gel quantitation. Some gel imagers are equipped with analysis software for simpler quantitation of samples in the gel, while others have cloud connectivity for data storage.

Figure 3. (A) Gel quantitation using fragments in known amounts. (B) Standard curve for gel quantitation.

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c. Analysis of sample purity, integrity, fragmentation, and synthesis efficiency

Gel electrophoresis may be employed to assess the purity and integrity of nucleic acid samples after extraction from their sources, as well as the success of sample fragmentation, and the percentage of full-length oligonucleotides after synthesis.

  • Genomic DNA that contaminates RNA samples, and vice versa, may be detected using gel electrophoresis to examine sample purity (Figure 4A). The detection of contaminating species will depend on the sensitivity of the nucleic acid stain used, as well as the amount of contaminants.
  • Using gel electrophoresis, the integrity of total RNA after an extraction may be examined by assessing the relative intensities of 28S and 18S rRNAs, with a 2:1 ratio indicating intact RNA. The presence of smears, especially at lower molecular weights, is indicative of RNA degradation (Figure 4B).
  • Some protocols call for fragmentation of nucleic acid samples, such as in the preparation of sample input for chromatin immunoprecipitation (ChIP) and next-generation sequencing (NGS), to obtain fragment sizes appropriate for the next step. The efficiency of sample fragmentation can be examined by gel electrophoresis (Figure 4C).
  • Following synthesis, oligonucleotide products will contain full-length as well as truncated or failure sequences (i.e., n–1, n–2, etc.), due to coupling efficiency of synthesis that is <100% (learn more: Oligonucleotide synthesis). Electrophoresis is one way to differentiate full-length products from failure sequences based on the size and conformation of oligonucleotides of different lengths.

Figure 4. Gel electrophoresis in determining sample purity, integrity, and fragmentation. (A) Extracted genomic DNA and total RNA were analyzed on separate gels. The red arrows indicate contaminating RNA and genomic DNA, respectively. Contaminating RNA is detectable only at the beginning of electrophoresis (≤5 min run). (B) Two samples of purified RNA were assessed for integrity by electrophoresis and analysis of 28S and 18S rRNAs. (C) The efficiency of DNA fragmentation and distribution of fragmented DNA were determined on a gel. (M = molecular weight standard. RNA samples were run on denaturing gels.)

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d. Detection of sequences of interest in a mixture of samples

Gel electrophoresis is a critical part of the workflow in detecting target sequences in a pool of nucleic acids by probe hybridization (Figures 5, 6). Probes are single-stranded nucleic acids of known sequences that are designed to bind target sequences specifically via base complementarity.

  • For DNA fragment analysis, Southern blot and restriction enzyme fragment length polymorphism (RFLP) analysis are two well-known techniques in which electrophoresis is used to separate samples before target detection (Figure 5).

Figure 5. Southern and northern blots are used to detect specific DNA and RNA sequences, respectively, in a sample pool.

  • For RNA fragment analysis, northern blots and nuclease protection assays (NPA) also rely on probe hybridization to detect the sequences of interest. The northern blot follows the same workflow as that of the Southern, except the input is RNA (Figure 5). In the ribonuclease protection assay (RPA), RNA probes bind to the target sequences in the sample mixture. The remaining single-stranded RNAs, such as unbound probes and templates, and their overhangs, are then digested by ribonucleases like an RNase A/T1 mix. Bound probes and samples are then run on a gel for downstream detection and analysis of targets.

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e. Assessment of DNA conformation and nucleic acid–protein complex

As discussed in the section of electrophoresis considerations, plasmid DNA of the same sequence may show different electrophoretic mobilities, depending on conformation. This characteristic may be used to assess DNA conformation, as well as the level of intact plasmids after extraction.

Nucleic acid fragments, when bound to proteins, migrate more slowly in electrophoresis than when unbound. Formally known as the electrophoretic mobility shift assay (EMSA), the method based on this principle is commonly referred to as a gel shift or gel retardation assay, since the bound proteins shift or retard the nucleic acid fragments’ migration in gels (Figure 7). Therefore, the electrophoresis step of the assay can provide a “snapshot” of the equilibrium between bound and free DNA in the sample. For EMSA, a low–ionic strength buffer should be used in both the gel preparation and electrophoresis run to help stabilize the nucleic acid–protein complex during electrophoresis. 

Figure 7. Electrophoretic mobility shift assay.

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2. Preparative electrophoresis to purify nucleic acid samples

In preparative applications of nucleic acid gel electrophoresis, separated nucleic acids are purified from the gel matrix after electrophoretic analysis. Therefore, electrophoresis intended for gel purification is typically a preparation step for downstream applications. Logistically, gel preparative strategies may be performed in conjunction with the analytical approach.

a. Applications involving preparative electrophoresis

Preparative electrophoresis follows many molecular biology techniques and applications, most commonly: PCR; restriction digestion; oligonucleotide synthesis; and fragmentation, end modification, and ligation steps of next-generation sequencing (NGS).

  • PCR products and restriction enzyme–digested DNA may be purified from gels for downstream applications such as end modification, ligation, cloning, and sequencing. (Learn more: Restriction enzyme cloning, PCR cloning)
  • Following oligonucleotide synthesis, polyacrylamide gel electrophoresis is one of the primary methods for separating and purifying full-length oligonucleotides from salts and truncated sequences.
  • For Illumina and Ion Torrent NGS platforms, input samples are fragmented, end-modified, and ligated to adapters, as part of sequencing library preparation. Following these steps, DNA fragments in specific size ranges (e.g., 200–300 bp) are purified in a process called size selection, to remove not only low– and high–molecular weight fragments but also adapters, enzymes, and reagents from the prior steps. Size selection also helps ensure that sample inputs comprise fragments of uniform lengths for high-quality and consistent sequencing [1]. Gel electrophoresis is one method of size selection, since it is efficient to select fragments of specific sizes in a narrow range (Figure 8).

Figure 8. Distribution of fragment sizes before and after size selection.

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b. Overview of gel purification

Preparative electrophoresis is a critical step in the isolation and purification of nucleic acids from gels. Purification of the nucleic acid usually involves excising the desired sample band from the gel matrix, followed by extraction of the nucleic acid. When performing gel purification, care should be taken to:

  • Minimize the amount of gel material excised, to facilitate extraction
  • Prevent damage to nucleic acids during gel visualization

If a UV-excitable dye (e.g., ethidium bromide) is used for visualization, minimize the sample’s exposure to radiation, use a longer UV wavelength for excitation (e.g., 360 nm instead of 254 nm), and select epi-illumination over transillumination. Fluorescent dyes that can be excited by lower-energy blue light are better alternatives for sample visualization because they lessen sample damage from radiation.

Since agarose and polyacrylamide are different, purification strategies usually differ for the two gel matrices, as summarized below:

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i. Purification from agarose gels

After excision of the gel matrix with the desired band, agarose may be melted by heating. At a 1% gel concentration, standard agarose melts at >90°C and low melting point (LMP) agarose at >65°C. Due to its lower melting temperature, the use of LMP agarose can improve the integrity and yield of extracted nucleic acids. For a much gentler approach than heating, the enzyme agarase may be considered, to break down agarose chains into oligosaccharides. Agarase digestion requires the use of LMP agarose so that the agarase remains active at the low gelling temperature of LMP agarose.

After the agarose is melted or digested, nucleic acids may be extracted following one of several methods [2]. Phenol extraction followed by ethanol precipitation is a simple and efficient procedure for nucleic acid purification. Due to phenol’s toxicity and the requirement to treat it as organic waste, a popular method nowadays is to isolate nucleic acids by binding them to silica-based columns in the presence of chaotropic agents, then eluting them from the column (Figure 9).

Figure 9. Extraction of DNA from a gel matrix using silica-based columns.

Another very simple and fast method to isolate specific bands is using a special agarose gel with two rows of wells [3]. Samples are loaded into the top row, and as electrophoresis progresses, bands of desired sizes are retrieved from the bottom row (Figure 10).

Figure 10. Three simple steps for separation and isolation of DNA bands using specially designed precast agarose gels. Samples are loaded into the top row of wells, bands separate during the gel run, and individual bands are collected from the bottom row as they enter those wells.

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ii. Purification from polyacrylamide gels

Since polyacrylamide gels are formed by polymerization reactions, the gel matrix cannot be removed simply by heating. Nucleic acids are usually extracted from polyacrylamide gels by either “crush and soak” or electroelution methods [4] (Figure 11).

Figure 11. Extraction of nucleic acids from a polyacrylamide gel matrix.

  • In the crush and soak method, the excised gel band is crushed by pushing it through a syringe or using a small pestle. Freezing the gel slice can make it easier to crush. The crushed gel is allowed to soak in an elution buffer for 18–36 hours with gentle shaking. The eluted nucleic acids may then be ethanol precipitated for concentration and purification.
  • The electroelution method uses an electrical field to move nucleic acids out of the gel matrix. One simple approach is to place the excised band inside a dialysis membrane with a small molecular weight cutoff. The dialysis membrane containing the gel slice is then subjected to electrophoresis to elute nucleic acids out of the gel.

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In conclusion, nucleic acid gel electrophoresis has broad applications in a wide range of molecular biology workflows and techniques. Although its basic method largely remains unchanged since the 1970s, electrophoresis has proven to be a powerful technique for separation and analysis of nucleic acids, in applications ranging from restriction digestion to next-generation sequencing.

References

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