Nucleic Acid Electrophoresis Applications–Preparative and Analytical Electrophoresis

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.

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

• The polymerase chain reaction, or PCR, amplifies a single copy of a target sequence to millions of copies, in a few hours. Electrophoresis is performed following endpoint PCR, to confirm amplification of the target and its yield. (Learn more: PCR basics and applications)
• After restriction digestions, which are reactions with restriction enzymes to cleave specific sequences on DNA substrates, samples are run on a gel to determine the pattern of DNA cleavage (as well as the extent of completion of the digestion). (Learn more: Restriction enzyme basics and applications)
• In molecular cloning, a DNA fragment is inserted into a vector via a process called ligation. In some cases, electrophoresis may be performed after ligation to assess reaction efficiency (Figure 2). The ligated product is then used to transformcompetent cells of a cloning organism, like E. coli, for propagation, after which the colonies formed are screened to determine whether they carry the vector with the desired insert. PCR and restriction digestion, both of which often involve electrophoresis as part of the workflow, are common techniques used in colony screening. (Learn more: Molecular cloning basics, transformation basics, and colony screening methods)
• Reverse transcription is synthesis of DNA complementary to an RNA template (cDNA), by an enzyme called reverse transcriptase. After cDNA synthesis and removal of the RNA template, the product 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. (Learn more: Reverse transcription basics and applications)
• In vitro transcription is synthesis of RNA from a DNA template, by an RNA polymerase. After removal of the DNA template, the synthesized RNA may be run on a denaturing gel to determine success. (Learn more: In vitro transcription basics)

Top

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.

Top

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%. Electrophoresis is one way to differentiate full-length products from failure sequences based on the size and conformation of oligonucleotides of different lengths.

Top

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).

• 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.

Top

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. 

Top

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.

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).

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.

1. Head SR, Komori HK, LaMere SA et al. (2014) Library construction for next-generation sequencing: overviews and challenges. Biotechniques 56(2):61–4, 66, 68.
2. Moore D, Dowhan, D, Chory J et al. (2002) Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels. In: Ausubel FM et al. (editors) Current Protocols in Molecular Biology. Supplementary 59: Unit 2.6.
3. Gibson JF, Kelso S, Skevington JH (2010) Band-cutting no more: A method for the isolation and purification of target PCR bands from multiplex PCR products using new technology. Mol Phylogenet Evol 56:1126–1128.
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.