What are the applications of capillary electrophoresis?

The use of capillary electrophoresis (CE) in genetic analysis applications has largely replaced the use of gel separation techniques due to significant gains in workflow speed, throughput, and ease of use. Some of the advantages of CE compared to conventional polyacrylamide gel electrophoresis include:

• Ease of use—no gels to pour
• Reusability—the polymer matrix can be reused (product-dependent)
• Fast separation times
• Better resolution (single base pair)
• Automated reads—optical sequence reading
• Higher throughput—multiple capillaries can be used simultaneously
• Complete automation—a complete workflow (including CE) is available on automatic genetic analysis systems

CE enables a wide variety of applications for a broad range of research areas (Figure 1).

During capillary electrophoresis, products of the sequencing reaction enter the capillary as a result of electrokinetic injection. A high-voltage charge applied to the buffered sequencing reaction forces the negatively charged DNA fragments into the capillaries. The DNA fragments are separated by size due to the larger fragments migrating more slowly through the matrix. CE plays a central role in the overall workflow for both automated Sanger sequencing and fragment analysis (Figures 2 and 3).

Before reaching the positive electrode, the DNA fragments pass through a laser beam, which excites the dye labels and cause them to fluoresce. The dye signals are separated by a diffraction system, and a charge-coupled device (CCD) camera detects the fluorescence (Figure 4).

Because each dye emits light at a different wavelength when excited by the laser, all colors, and therefore loci, can be detected and distinguished in one capillary injection.

The fluorescence signals are converted to digital data, in a file format compatible with an analysis software application (Figure 5).

In some CE instruments like the Applied Biosystems SeqStudio instrument, a universal polymer allows for flexibility to perform Sanger sequencing and fragment analysis on the same instrument run with the same cartridge.

Since CE plays a central role in both automated Sanger sequencing and fragment analysis workflows, it enables applications for both.

Sanger sequencing is the gold standard of sequencing technology: it provides high accuracy, long-read capabilities, and flexibility to support a diverse range of applications. Sanger sequencing is mostly known for DNA sequencing applications, but also supports applications in RNA sequencing and epigenetic analysis.

• De novo DNA sequencing—De novo sequencing is the term used to describe initial sequence analysis performed to obtain the primary genetic sequence of a particular organism. A detailed genetic analysis of an organism is possible only after de novo sequencing has been performed. For de novo sequencing using CE, the target DNA is fragmented and cloned into a viral or plasmid vector.
• Targeted DNA sequencing—Identifying heterozygous base positions or small insertions or deletions in genomic DNA is often employed to locate mutations or polymorphisms in diploid organisms, detect genetic rearrangements, and uncover rare variants.
• NGS confirmation—NGS analyses have revolutionized our understanding of biological processes. For critical studies, data obtained from next-generation sequencing systems should be confirmed. Sanger sequencing by CE is an ideal orthogonal technology for verification of NGS base calls.
• Genome editing confirmation—Sanger sequencing can be used to determine the efficiency of CRISPR-mediated genome editing in primary transformed cell cultures, and to determine successful editing events in secondary clones.
• Microbial sequencing—Microbial sequencing is performed for a number of reasons, such as microbial identification, environmental monitoring, pathogen detection, and routine testing of materials for bacterial contamination.
• Mitochondrial sequencing—Mitochondrial DNA sequencing is a useful tool for studying human diseases such as diabetes, certain cancers, and mechanisms of aging. It is also used in population genetics and biodiversity assessments, as well as forensics. Targeted mitochondrial sequencing can be used to detect mutations present in some copies of the mitochondrial genome (heteroplasmic mutations).
• Viral Vector QC—Sequencing of ITR regions before producing viral particles, assessing purity, measuring capsid and vector genome titers, and verifying that the DNA sequence matches the intended design.

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DNA fragment analysis enables a multitude of applications from genotyping to bacteria identification and from plant screening to gene expression profiling. See how you can adapt these applications to broaden the possibilities for your research.

• Short tandem repeat (STR) analysis in human sample identification—STR genotyping is an important tool for verifying the authenticity of human cell lines, quality control of stored human tissues and fluids, and assessing the nature of known mixtures. An STR analysis workflow that uses CE is a simple, economical method and gold standard for studying human samples.
• Microsatellite marker analysis—Microsatellite markers are polymorphic DNA loci containing repeated nucleotide sequences, typically 2 to 7 nucleotides per repeat unit. The length of the repeat is the same for the majority of the repeats within an individual microsatellite locus, but the number of repeats for a specific locus may differ, resulting in alleles of varying length that can be subjected to fragment analysis by capillary electrophoresis. Because they are inherited in Mendelian fashion, analysis of length variations is widely accepted for applications such as microsatellite instability (MSI).
• Quantitative fluorescence PCR (QF-PCR)—Relative fluorescent quantitation or quantitative fluorescence PCR is a technique used in a variety of fragment analysis applications that require accurate peak height or peak area comparisons across multiple samples. Applications that utilize this technique include screening for loss of heterozygosity (LOH), aneuploidy assays, and detecting large chromosomal deletions.
• Restriction fragment length polymorphisms (RFLP) analysis—DNA sequence polymorphisms display different migration profiles from wild-type fragment patterns when DNA is digested with restriction fragments and separated by size. T-RFLP (terminal-RFLP) analysis is a culture-independent RFLP method (only terminal fragments are labeled and detected) used to study highly complex microbial populations, based on variations in 16S rRNA (ribosomal RNA). This technique allows researchers to examine complex communities without the need for any genomic sequence information. This method is rapid, sensitive, reproducible, and enables the study of bacterial populations in diverse natural habitats, and the organisms’ responses to changes in environmental or physiological parameters.
• Amplified fragment length polymorphism (AFLP) analysis—AFLP analysis is a technique used to detect polymorphisms in DNA when no information about the genome is known. Because it can be used to interrogate multiple regions simultaneously, it is often used to identify genetic variation in strains or closely related species of plants, fungi, animals, and bacteria.
• Single-stranded conformation polymorphism (SSCP) analysis—SSCP analysis is a widely used screening method for identifying genomic variants in a large number of samples and in a broad range of organisms, from microorganisms to humans. SSCP analysis detects sequence variations (single-point mutations and other small-scale changes) through electrophoretic mobility differences. When subjected to nondenaturing (or partially denaturing) electrophoresis, DNA that contains a sequence mutation (even a single base pair change) and the wild-type sequence have measurable mobility differences.
• Single-nucleotide polymorphism (SNP) genotyping—SNP genotyping provides a measure of genetic variation. SNPs are one of the most common types of genetic variation

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From research to single-gene to small gene panels, CE can help achieve research goals across a diverse set of applications.