How to Conduct Sanger Sequencing

Sanger sequencing is a method that yields information about the identity and order of the four nucleotide bases in a segment of DNA. Also known as the “chain-termination method”, it was developed in 1977 by Frederick Sanger and colleagues, and is still considered the gold standard of sequencing technology due to its relative simplicity, accuracy, and reliability.

In order to provide a sequence, Sanger sequencing makes use of chemical analogs of the nucleotide bases. The analogs, called dideoxyribonucleotides (ddNTPs), are missing the hydroxyl group that is required for extension of a DNA polynucleotide chain. By mixing dye-labeled ddNTPs and template DNA in a PCR-based cycle sequencing reaction, strands of each possible length are produced when the ddNTPs get randomly incorporated and terminate the chain.

There are six steps in the Sanger sequencing workflow from sample to data (Figure 1). When starting with a low amount of DNA, the first step is PCR amplification of the target to ensure that there will be enough template for the sequencing reaction to proceed, followed by a clean-up step to remove excess primers. The PCR products are then used in a cycle sequencing reaction to generate chain-terminated fragments. This is followed by another clean-up step to remove unincorporated ddNTPs that could affect the genetic analyzer’s ability to detect the dyes. The fragments are then separated by capillary electrophoresis (CE), and the sequence is read using data analysis software.

Sanger sequencing targets a region of interest in template DNA using an oligonucleotide sequencing primer, which binds upstream of the target that is to be sequenced (there must be an area of known sequence close to the target). In contemporary usage, purified PCR products most often serve as the sequencing template, but the flexibility of the technique allows for different types of single- or double-stranded DNA to be used (another common template being bacterial plasmid DNA).

For general guidelines on DNA isolation and preparing single- and double-stranded templates, see the DNA Sequencing by Capillary Electrophoresis Chemistry Guide.

Whether for PCR or cycle sequencing, it is important to design primers that will bind their target sequence during thermal cycling (for general guidelines on primer design, see [1]). There are several free online tools available that can assist with primer design.

It is necessary to extract the DNA from the source material prior to beginning the Sanger sequencing workflow. The quality of the DNA to be sequenced can be significantly affected by characteristics of the sample itself and the method chosen for nucleic acid extraction and purification. Ideal methods will vary depending on the source or tissue type, how the sample was obtained from its source, and how the sample was handled or stored prior to extraction. Common sources include bacterial colonies, tissue, blood or plasma, cells, and plant material. DNA can be extracted using a kit or following a lab-developed protocol. Note that there are different ways to extract DNA, each with certain advantages and unique pitfalls to avoid. Methods include:

• Organic chemical extraction (phenol-chloroform) followed by ethanol precipitation—relatively inexpensive and time-tested, but involves harmful reagents
• Inorganic chemical extraction (proteinase K and salt)—relatively inexpensive but prone to carryover of impurities
• Silica column–based extraction—many commercially available kits are spin column–based
• Magnetic bead–based extraction—easily automated

Follow your chosen method’s instructions for DNA extraction and subsequent purification.

Depending on the amount of DNA recovered, primers may be used to amplify the extracted DNA via PCR. There are several PCR master mixes available that contain a high-performance DNA polymerase, salts such as magnesium chloride (MgCl₂), buffer, and nucleotides, to simplify setting up PCR reactions. The other reaction components required are template DNA, primers, and nuclease-free water.

Once the reactions are set up, they are run on a thermal cycler. A typical PCR program includes initial denaturation followed by repeated cycles of denaturation, annealing, and extension steps, with a final hold at 4°C.

Once the template DNA has been amplified by PCR, clean-up is required to remove unincorporated primers; any leftover primers can interfere with the strand-specific cycle sequencing reactions that will follow. There are several methods for removing unincorporated primers:

• Ethanol/EDTA precipitation—inexpensive, produces a single band, but can cause loss of small PCR products
• Spin columns—many commercially available kits are spin column–based
• Enzymatic reaction—hydrolyzes primers and unincorporated nucleotides in a single step

The sequencing reaction is then run on the thermal cycler, using the PCR products as a template. By mixing labeled ddNTPs together with dNTPs, a single primer, polymerase, and a DNA template, chain-terminated, end-labeled fragments representing each base in a segment of DNA can be generated and subsequently read as a sequence. This is known as cycle sequencing, since the fragments are generated in cycles of denaturing, annealing, and extension. 

For this step, a master mix containing a high-performance DNA sequencing polymerase, salts, and nucleotides should be used to simplify the cycle sequencing reaction setup. Note that unlike in PCR, which uses two primers, only a single primer is used to generate single-stranded fragments during each cycle of sequencing.

The program on the thermal cycler will be similar to that of the PCR.

A sequencing clean-up step is then performed prior to capillary electrophoresis. This is different from the PCR reaction clean-up; this step is needed to remove unincorporated ddNTPs from the reaction, as well as salts and other contaminants. If the fluorescent ddNTPs are not removed, their fluorescent signals interfere with the signals from the desired fragments. Common cycle sequencing clean-up methods include:

• Ethanol/EDTA precipitation of single-stranded DNA
• Matrices that bind and remove small molecules like ddNTPs
  • Spin-column matrix
  • Matrix in the form of an additive that can be applied directly to the reaction
• Silica-based products that bind single-stranded DNA

Capillary electrophoresis (CE) is an electrophoresis technique that utilizes small glass capillaries filled with polymer. Each sequencing reaction is run in a single, dedicated capillary. Capillaries are provided in bundles, collectively referred to as an array, so that many reactions can be processed in parallel. The array fits into an instrument called a genetic analyzer, which provides the voltage, temperature, buffer, and polymer required for electrophoresis, and holds 96-well plates that contain the samples to be sequenced.

The electrophoresis within these capillaries separates the labeled chain-terminated fragments by length. CE can separate these molecules with single-nucleotide resolution.

As the fragments migrate through the capillary, a laser excites the fluorescent label on the ddNTP incorporated at the end of each terminated chain. Because each of the four ddNTPs is labeled with a different color, the signal emitted by each excited nucleotide corresponds to a specific base.

Once the run is finished, the instrument will generate an .ab1 file. Several software packages are available that can convert the fluorescent peaks of each nucleotide into a sequence (Figure 2).

In summary, Sanger sequencing can be a powerful tool for determining the sequence of a small number of genes, the basics of which are primer design, DNA template preparation, PCR and clean-up, cycle sequencing and clean-up, CE, and data analysis.

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1. Lorenz TC (2012) Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies. J Vis Exper 63:e3998.