Scientist pipetting from a vial with a desktop in the background portraying DNA

Nucleic acid gel electrophoresis is widely used in molecular biology research. Gel electrophoresis is a proven and effective method to separate, resolve, and quantitate nucleic acid molecules. The applications of gel electrophoresis can be grouped as analytical and preparative. Analytical gel electrophoresis is used to examine experimental outcomes, while preparative gel electrophoresis is used to isolate and purify samples for downstream processes.

Analytical applications of gel electrophoresis to determine experimental outcomes

The analytical applications of gel electrophoresis examine the 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, mobility, and hybridization, as described below.

Using gel electrophoresis to analyze enzymatic synthesis, digestion, and cloning experiments

Gel electrophoresis of nucleic acids is commonly used to determine the success and efficiency of molecular biology experiments. Figure 1 summarizes the uses of gel electrophoresis to determine experimental outcomes.

Figure 1. Uses of gel electrophoresis to determine experimental outcome. Applications which use gel electrophoresis include polymerase chain reaction (PCR), restriction digestion, ligation, colony screening, reverse transcription, and in vitro transcription.

  • In polymerase chain reaction or PCR, electrophoresis is performed to confirm amplification of the target DNA sequence and its yield following endpoint PCR. 
  • In restriction digestion, researchers use electrophoresis to evaluate the cleavage pattern of DNA sequences treated with restriction enzymes. Electrophoresis also reveals the extent of completion of restriction digestion. 
  • In molecular cloning, electrophoresis may be performed to assess efficiency of ligation and transformation. 
  • Testing for ligation efficiency ensures that the target DNA sequence is integrated into the vector DNA. (Figure 2). The ligated product is then used to transform competent cells of a cloning organism such as E. coli.
  • Screening colonies of transformed cells to detect the presence of desired DNA insert. Electrophoresis is part of PCR and restriction digestion workflows; both techniques are used in colony screening.
  • In cDNA synthesis, the freshly synthesized cDNA, from which the template RNA is removed, may be run on a denaturing gel to assess reaction efficiency. The method is most useful when a known sequence or length of RNA is reverse transcribed. 
  • In in vitro transcription, the newly synthesized RNA may be run on a denaturing gel to determine the success of transcription. 

Using gel electrophoresis to quantitate nucleic acid samples

Electrophoresis may be used to quantitate the mass of DNA or RNA fragments of interest using a standard, or ladder. Quantitation of nucleic acids by electrophoresis is more reliable compared to widely used spectrophotometric quantitation methods. Presence of contaminants, such as nucleotides and primers, interfere with the outcome of the spectrophotometric quantitation method.

Quantitation applications of gel electrophoresis are: 

  • To estimate the quantity of nucleic acid fragments in a sample, the intensity of the band of interest is compared with the intensity of a matching band in the ladder (Figure 3A). 
  • For accurate quantification, a standard curve for band intensity for varying dilutions of ladder is plotted. The quantity of DNA or RNA in the sample can be inferred from the relative band intensities of the ladder (Figure 3B).
  • Staining nucleic acids with a fluorescent dye that has high sensitivity and wide dynamic range improves the accuracy of quantification. The dyes bind to DNA or RNA and fluoresce, which is typically captured by gel imagers. Gel imagers are typically equipped with analysis software that are available as desktop applications or for quantitation of nucleic acid fragments in the gel.

Figure 3. Quantitation of nucleic acid samples by gel electrophoresis. (A) Gel quantitation using fragments in known amounts to compare to a sample of unknown amount. (B) Standard curve for gel quantitation using the samples of known amounts to determine the concentration of the sample of unknown amount.

Using gel electrophoresis to analyze 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.

  • Gel electrophoresis can be used to evaluate the purity of nucleic acid samples by detecting contaminants, such as genomic DNA in RNA samples (Figure 4A). Factors that affect the ability to detect contaminants include the sensitivity of the stain and the amount of contaminants present.
  • The integrity of total RNA samples can be assessed using gel electrophoresis by comparing the relative intensities of 28S and 18S rRNAs (Figure 4B). A 2:1 ratio indicates intact RNA, while smears at lower molecular weights indicate degradation.
  • 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).
  • Gel electrophoresis can also be used to differentiate full-length oligonucleotide products from truncated or failure sequences, which are often present due to less than 100% coupling efficiency during synthesis.

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.)

Using gel electrophoresis to detect sequences of interest in a mixture of samples

Gel electrophoresis is a critical part of the workflow to detect 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.

Figure 5. Southern and northern blots are used to detect specific DNA and RNA sequences, respectively, in a sample pool. Workflow steps begin with sample preparation, then gel electrophoresis followed by blotting, probe hybridization, and visualization.

  • For RNA fragment analysis, northern blots and nuclease protection assays (NPA) also rely on probe hybridization to detect the sequences of interest (Figures 5, 6). The northern blot follows the same workflow as that of the southern, except the input is RNA. In the ribonuclease protection assay (RPA), RNA probes bind to the target sequences in the sample mixture. Bound probes and samples are then run on a gel for detection and analysis of targets.

Using gel electrophoresis to assess DNA conformation and nucleic acid-protein complexes

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. This principle is used in the electrophoretic mobility shift assay (EMSA), also known as the gel shift or gel retardation assay, which allows for the detection of protein-DNA interactions in a sample (Figure 7). The electrophoresis step provides a "snapshot" of the equilibrium between bound and free DNA in the sample. To optimize the assay, it is important to use a low-ionic strength buffer in both the gel preparation and electrophoresis run to stabilize the nucleic acid-protein complex.

Preparative electrophoresis to purify nucleic acid samples

Preparative gel electrophoresis includes purification of separated nucleic acids from the gel matrix after standard electrophoresis. Generally, the band of interest is excised and processed to retrieve the DNA or RNA fragments. When using specialty precast gels such as CloneWell II gels, the DNA from the band of choice is collected at the recovery well.

The gel-purified nucleic acid fragments are typically used for downstream applications. Often, electrophoresis performed for analytical purposes serves as a precursor for preparative electrophoresis.

Preparative gel electrophoresis applications

Preparative electrophoresis is the subsequent step in many molecular biology techniques and applications, including PCR, restriction digestion, oligonucleotide synthesis, fragmentation, end modification, and ligation steps of next-generation sequencing (NGS).

Learn more: Restriction enzyme cloning
Learn more: PCR cloning

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 are purified by size selection, to remove low- and high-molecular weight fragments, adapters, enzymes, and reagents from the prior steps. Size selection also ensures 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).

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.

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