Sanger sequencing by capillary electrophoresis is a powerful technique routinely used in studying gene expression, identifying low-allelic fraction variants, and validating NGS approaches. Developed by Frederick Sanger and colleagues in 1977, this method uses fluorescently-labeled chain-terminating dideoxynucleotide triphosphates (ddNTPs) and subsequent size separation by capillary electrophoresis to determine the precise order of nucleic acid residues in a DNA
molecule. While the advent of high-throughput sequencing technologies, such as Next-Generation Sequencing (NGS), facilitates the large-scale sequencing of DNA molecules in parallel, the robustness and accuracy (99.99%) afforded by Sanger sequencing remains best suited for medium to low throughput sequencing projects.
Sanger Sequencing Workflow
Sanger sequencing, which is based on the principle and biochemistry of DNA replication, follows two basic steps:
- PCR using fluorescent chain-terminating dideoxynucleotides (ddNTPs)
- Size separation and analysis by capillary electrophoresis
Chain Termination PCR
In the first step, chain-termination PCR is used to replicate the DNA template of interest. This modified version of PCR uses all the same components as the standard PCR method, but with one key difference, the addition of fluorescently labeled dideoxynucleotides (ddNTPs) to the reaction mixture, such as MagaDye™ ddNTPs
from AAT Bioquest (Table 1). Structurally, ddNTPs differ from deoxynucleotides (dNTPs) in that the 3' carbon of the deoxyribose sugar is attached to a hydrogen molecule (-H) instead of a hydroxyl group (-OH). This absence of the 3'-OH group interrupts the condensation reaction during DNA replication and prevents further extension of the amplicon. The result of many of these reactions is a large number of fluorescent DNA fragments terminated at random lengths.
Size Separation and Analysis by Capillary Electrophoresis
In the second step, the fluorescent chain-terminated DNA fragments are separated and identified via capillary electrophoresis (CE). In this method, DNA fragments are electrokinetically injected into very thin polymer-filled capillaries and, under the influence of an electric field, migrate through the capillary array. The fragments are separated by size, with the smallest fragments moving fastest. As they move past the detection window, a laser excites the fluorescent tags in each band, and the emitted light is spectrally separated and recorded by a CCD camera. Instrument software analyzes the raw data and outputs it as a chromatogram illustrating the fluorescent peak of each nucleotide along the length of the DNA template.
An illustration of Sanger sequencing by capillary electrophoresis (figure made in BioRender).
Fluorescent ddNTPs for Sanger Sequencing
MagaDye™ Sanger Sequencing ddNTPs
The MagaDye™ 4 Color Sanger Sequencing Terminator Kit, developed by AAT Bioquest, is a streamlined solution for use in any sequencing and genotyping application. The four distinct fluorescent ddNTPs are readily detected using the 488 nm laser line and emit at four different fluoresce colors with λmaxima centered at 536 nm, 561 nm, 588 nm, and 613 nm. Each ddNTP provided in this kit is supplied as 5 nmoles. The four MagaDye™ fluorescent ddNTP terminators are also available as standalone reagents - MagaDye™ 535-ddGTP, MagaDye™ 561-ddATP, MagaDye™ 588-ddTTP and MagaDye™ 613-ddCTP (Table 1).
Normalized excitation (left) and emission (right) spectra for MagaDye™ ddNTP terminators.
Table 1. Sanger sequencing terminator kit and dye-labeled ddNTPs suitable for Sanger sequencing
|MagaDye™ 4 Color Sanger Sequencing Terminator Kit||498||536/561/588/614||5 nmoles||17068|
|MagaDye™ 535-ddGTP||503||536||5 nmoles||17063|
|MagaDye™ 535-ddGTP||503||536||50 nmoles||17067|
|MagaDye™ 561-ddATP||498||561||5 nmoles||17062|
|MagaDye™ 561-ddATP||498||561||50 nmoles||17066|
|MagaDye™ 588-ddTTP||498||588||5 nmoles||17061|
|MagaDye™ 588-ddTTP||498||588||50 nmoles||17065|
|MagaDye™ 613-ddCTP||498||614||5 nmoles||17060|
|MagaDye™ 613-ddCTP||498||614||50 nmoles||17604|
|2-Aminoethoxypropargyl ddCTP|| || ||1 µmoles||17080|
Table 2. Building Blocks for Preparing Fluorescent Conjugates in NGS