Capillary Electrophoresis

From slab to capillary

Capillary electrophoresis (CE) separates DNA by size like a gel, but inside a fused-silica capillary about 50 µm across, filled with a liquid sieving polymer instead of a cast gel ¹. Because the narrow capillary sheds heat well, it can run at high voltage: fast, sharp separations with single-base resolution. Instruments run 8 to 96 capillaries at once and refill the polymer automatically.

Samples (dye-labeled DNA in formamide, denatured) are loaded by electrokinetic injection: a brief voltage pulls DNA into the capillary tip. Salt competes with DNA for injection, so salty or unpurified samples load poorly.

The electropherogram

Near the capillary’s end, a laser excites dye-labeled fragments as they pass a window, and a detector records their emission. Small fragments arrive first. The plot of fluorescence (RFU) against time - converted to size - is the electropherogram: each fragment is a peak ¹.

Dyes and the size standard

The detector separates several dye colors at once, so different markers (or different bases, in sequencing) are labeled with different dyes and run together in one capillary.

One color is reserved for an internal size standard - fragments of known size added to every sample. Migration varies between capillaries and runs, so the software sizes each sample peak against the standard in its own capillary ¹. Sizes come out to a fraction of a base; reproducibility depends on the same polymer, temperature, and standard.

Pull-up is an artifact where a very strong peak in one color bleeds into another color’s channel at the same position; overloading makes it worse.

Two clinical readouts

Sanger sequencing

The sequencing reaction makes fragments ending at every position, each tagged with one of four dye-labeled terminators, one color per base. CE lines them up by size, one base apart, and the color of each successive peak gives the sequence ². A heterozygous base shows two overlapping peaks of different colors at one position. (Chemistry in the amplification and sequencing course.)

Fragment analysis

Here the goal is size, not sequence: dye-labeled PCR products are sized against the standard ¹.

Fragment-analysis artifacts to know: stutter (a peak one repeat shorter, from polymerase slippage, usually well under the parent peak), +A (Taq’s extra adenine adding a base), and pull-up between colors.

What to take from this

CE separates labeled DNA by size in a polymer-filled capillary at single-base resolution, detecting fragments with a laser as they pass. The electropherogram plots RFU against size, calibrated by an internal size standard in its own dye in every capillary. Sanger sequencing reads four colors one base apart; fragment analysis sizes products for STRs, repeat expansions, MSI, and clonality. Recognize stutter, +A, and pull-up.