Gel Electrophoresis Applications: Preparative and Analytical Electrophoresis

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. 

Learn more about restriction enzyme basics and applications
Learn more about in vitro transcription basics 

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.

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.

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.

• 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 (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.

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