Actively helping customers, employees and the global community during the coronavirus SARS-CoV-2 outbreak.  Learn more >>

Sanger Sequencing

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:
  1. PCR using fluorescent chain-terminating dideoxynucleotides (ddNTPs)
  2. 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

Product
Ex (nm)
Em (nm)
Unit Size
Cat No.
MagaDye™ 4 Color Sanger Sequencing Terminator Kit498536/561/588/6145 nmoles17068
MagaDye™ 535-ddGTP5035365 nmoles17063
MagaDye™ 535-ddGTP50353650 nmoles17067
MagaDye™ 561-ddATP4985615 nmoles17062
MagaDye™ 561-ddATP49856150 nmoles17066
MagaDye™ 588-ddTTP4985885 nmoles17061
MagaDye™ 588-ddTTP49858850 nmoles17065
MagaDye™ 613-ddCTP4986145 nmoles17060
MagaDye™ 613-ddCTP49861450 nmoles17604
2-Aminoethoxypropargyl ddCTP 1 µmoles17080

Table 2. Building Blocks for Preparing Fluorescent Conjugates in NGS

Product
Unit Size
Cat No.
5-Propargylamino-3'-azidomethyl-dCTP50 nmoles17091
5-Propargylamino-3'-azidomethyl-dUTP50 nmoles17093
7-Deaza-7-Propargylamino-3'-azidomethyl-dATP50 nmoles17090
7-Deaza-7-Propargylamino-3'-azidomethyl-dGTP50 nmoles17092