Capillary Electrophoresis

Overview

Capillary electrophoresis (CE) rests on the same principle as the slab-gel separation covered in the gel-electrophoresis lesson: nucleic acids carry a uniform negative charge from their phosphate backbone, so an electric field drives them through a sieving medium and a fragment’s migration depends on its size. What CE changes is the format. Instead of a thick slab of agarose or polyacrylamide, the separation happens inside a narrow glass capillary filled with a flowable polymer solution. That single change in geometry is what gives CE its defining advantages — single-base resolution, automation, and on-line detection — and it is why CE became the workhorse readout for clinical sizing and sequencing assays ¹.

This lesson assumes the gel-electrophoresis fundamentals (charge-to-size separation, sieving matrices, why smaller fragments move faster) and does not re-teach them. The focus here is the capillary format and the clinical readouts it enables.

From slab gel to capillary

A capillary is a hair-thin fused-silica tube, typically tens of centimeters long, filled with a viscous, replaceable polymer that acts as the sieving matrix in place of a cast gel. The capillary’s small diameter is the key. A thin tube dissipates heat efficiently, so CE can run at much higher voltages than a slab gel without the matrix overheating and distorting the separation. Higher field strength sharpens the separation of fragments that differ by only a single nucleotide ².

The workflow is highly automated. A sample is injected into one end of the capillary electrokinetically — a brief voltage pulse pulls charged molecules into the tube — and the separation voltage is then applied. Because the polymer is pumped in fresh for each run, the instrument can process sample after sample without an operator pouring and loading gels. This combination of high resolution and hands-off operation is what moved size-based separation from a manual gel technique to an instrument readout ¹.

On-line fluorescence detection and the electropherogram

In a slab gel the separated bands are visualized after the run by staining the whole gel. CE instead detects fragments during the run, as they migrate. Near the far end of the capillary is a fixed detector window. Fragments are labeled with fluorescent dyes; as each population passes the window, a laser excites the dye and the emitted light is recorded. Because all fragments travel the same fixed distance to the detector, smaller fragments — which migrate faster — reach the window first, and larger fragments arrive later ¹.

The instrument plots detected fluorescence against time. Each cluster of identically sized fragments produces a peak, and the sequence of peaks along the time axis is an electropherogram: a trace of peaks ordered from smaller (earlier) to larger (later) fragments. The electropherogram is the CE equivalent of a lane of bands on a gel, but with far finer resolution and a quantitative signal for each peak.

Electropherogram (fluorescence vs. migration time / fragment size)

signal
  |                  .                 single peaks = discrete
  |        .         |         .       fragment sizes
  |        |    .    |    .    |
  |   .    |    |    |    |    |    .
  |___|____|____|____|____|____|____|____> migration time
   small ----------------------------> large
       (faster)                  (slower)

The horizontal position of a peak reflects how long the fragment took to reach the detector, which corresponds to its size; the height (or area) of a peak reflects how much of that fragment is present. Converting a peak’s position into an actual size in bases requires a known reference run in the same lane, which is the role of the internal size standard described below ¹.

Multi-dye multiplexing and the internal size standard

Two related ideas let one capillary run report several kinds of information at once: spectral multiplexing and an internal size standard.

Multi-dye multiplexing. The detector can distinguish several fluorescent dyes that emit at different wavelengths (different colors). Because the instrument records color as well as migration time, fragments labeled with different dyes can travel through the same capillary simultaneously and still be read as separate channels. This is what allows a single run to carry, for example, several differently labeled targets plus a reference set, each resolved by its own color ¹.

Internal size standard. Migration time alone is not a reliable measure of size — it drifts run to run with temperature, polymer lot, and field conditions. To convert migration time into an accurate fragment size in bases, a set of DNA fragments of known sizes, labeled with a dye reserved for this purpose, is added to every sample. These standard fragments run in the same capillary as the sample, in their own color channel. The instrument fits a sizing curve to the known standard peaks and then reads each sample peak’s size off that curve. Because the standard travels under identical conditions as the sample, run-to-run variation is corrected within each run ¹.

Clinical CE readouts

CE underlies two broad families of molecular tests. They share the capillary, the dyes, and the size standard, but differ in what the peaks mean.

Sanger sequencing readout

In Sanger (chain-termination) sequencing, the reaction generates a nested set of DNA fragments that each end in one of four fluorescently labeled terminators — one color per base. CE separates this ladder at single-base resolution, and the detector reads the color of each successive peak. Reading the colors in migration order reconstructs the base sequence. The reaction chemistry that produces the four-color ladder is covered in the Sanger sequencing lesson and is not repeated here; the point for CE is that the capillary provides the single-base separation and color-by-color readout that make the method work ³.

Fragment analysis and genotyping

In fragment analysis, the goal is not to read a sequence but to size one or more fluorescently labeled PCR products precisely. Primers are labeled with dyes, the PCR products are mixed with the internal size standard, and CE reports each product’s size in bases against that standard. Differences in product size correspond to differences in genotype. Two important clinical applications are:

• Short tandem repeat (STR) profiling. STR markers are short repeated sequences whose repeat count varies between individuals. PCR products spanning each STR are sized by CE, and the resulting pattern of peak sizes forms a profile. This is the basis of specimen identity testing and of monitoring the mix of donor and recipient cells after transplant; those applications are developed in their own lessons by topic ¹.

• Sizing repeat expansions. Some disorders are caused by an expanded number of repeated trinucleotides in a gene. Because more repeats make a larger PCR product, CE sizing can estimate how many repeats are present by measuring product size against the standard. The disease biology of these trinucleotide-repeat disorders is covered in its own lesson by topic; here the relevant idea is that repeat number maps to fragment size, which CE measures ¹.

In both applications the internal size standard is what makes the readout quantitative and comparable across runs, and multi-dye multiplexing is what lets several markers (and the standard) share one capillary.

Summary

Capillary electrophoresis keeps the charge-based, size-dependent separation of gel electrophoresis but moves it into a thin, polymer-filled capillary run at high voltage. The format yields single-base resolution, automated sample-to-sample operation, and on-line fluorescence detection that produces an electropherogram of peaks ordered by size. Multi-dye multiplexing and an internal size standard turn raw migration into accurate, multi-channel size measurements. On this single platform sit the major clinical readouts: four-color Sanger sequencing and fragment analysis for STR profiling and repeat-expansion sizing.