Sanger sequencing, also called dideoxy sequencing or the chain-termination method, is a technique used to determine the order of nucleotides within a DNA molecule.

The method was introduced in 1977 by Frederick Sanger, Steven Nicklen and Alan Coulson. Its central principle is based on nucleotide analogues called dideoxynucleotides, which stop DNA-chain elongation when incorporated during DNA synthesis.

Instead of generating only one complete copy of a DNA template, the sequencing reaction produces many DNA fragments terminating at different nucleotide positions.

Determining the size and terminal nucleotide of these fragments allows the sequence of the DNA to be reconstructed.

Sanger sequencing played an important role in the development of modern genomics and was extensively used during large genome-sequencing projects before massively parallel sequencing technologies became available.

Today, it remains highly useful for targeted molecular biology applications where researchers need accurate sequence information from a relatively small number of defined DNA regions.

The main purpose of Sanger sequencing is to determine or verify the nucleotide sequence of a specific DNA region.

Because Sanger sequencing is a targeted approach, it is particularly useful when researchers do not need to sequence thousands or millions of DNA fragments simultaneously.

Sanger sequencing works through primer-directed DNA synthesis combined with controlled chain termination.

DNA polymerase normally extends DNA by adding nucleotides to the 3′ end of a growing DNA strand.

During a Sanger sequencing reaction, normal deoxynucleotides are present together with a smaller proportion of chain-terminating dideoxynucleotides.

When a normal dNTP is incorporated, DNA synthesis continues.

When a ddNTP is incorporated, the growing DNA strand cannot be extended further.

Because termination occurs at different positions among the many DNA molecules present in the reaction, a collection of DNA fragments of different lengths is generated.

These fragments can then be separated according to size, and the terminal nucleotide of each fragment can be identified.

The exact composition of sequencing reactions depends on the chemistry and platform being used, but the underlying method requires several fundamental components.

The biochemical difference between dNTPs and ddNTPs is central to the Sanger sequencing method.

This deliberate interruption of DNA-chain elongation is why Sanger sequencing is often called chain-termination sequencing.

The following workflow summarizes the major stages involved in a typical Sanger sequencing experiment. Exact reaction conditions depend on the chemistry, instrument and sequencing service being used.

Because ddNTP incorporation occurs at different positions across a population of DNA molecules, fragments can be produced that terminate after successive nucleotide positions.

Example population of terminated fragments:

5′-A
5′-AT
5′-ATG
5′-ATGC
5′-ATGCA
5′-ATGCAT
5′-ATGCATG
...

Shortest fragment
        ↓
Longer fragments
        ↓
DNA sequence reconstruction

In an actual sequencing experiment, a large collection of fragments is produced and separated with enough resolution to distinguish DNA molecules differing by approximately one nucleotide in length.

Capillary electrophoresis separates the chain-terminated DNA fragments according to their length.

Shorter DNA fragments generally migrate through the separation matrix more rapidly than longer fragments.

As fragments pass through the detection region, their fluorescent terminal nucleotide is identified. The order of detected signals can then be converted into a DNA sequence.

A Sanger sequencing chromatogram , also called an electropherogram, is a graphical representation of the fluorescent signals recorded during fragment analysis.

1. Read the sequence from left to right

In a typical software display, successive base calls are shown from left to right starting from the region adjacent to the sequencing primer.

2. Identify individual nucleotide peaks

Each well-resolved major peak represents a nucleotide call:

• A - Adenine
• C - Cytosine
• G - Guanine
• T - Thymine

3. Examine peak separation

A high-quality sequencing region generally contains distinct individual peaks with relatively low background signal.

4. Do not assume universal peak colors

Different analysis programs may use different colors for A, C, G and T. Nucleotide identity should therefore be determined using the software labels or legend.

5. Examine overlapping peaks

Multiple signals at the same nucleotide position may indicate mixed DNA templates, sequence heterogeneity or other sources of ambiguous sequencing signal.

6. Inspect the beginning and end carefully

Sequence quality is not necessarily constant across the entire read. Regions close to the sequencing primer and regions farther into a long read may sometimes be less clearly resolved.

Classical Sanger sequencing used four electrophoretic lanes corresponding to the four nucleotides: A, C, G and T.

A        C        G        T


                  ●


         ●


●


                           ●


                  ●


         ●


↑
Read from bottom to top

Shortest DNA fragment first

The shortest fragments migrate farthest through the gel and therefore appear closest to the bottom.

Starting at the lowest band, identify whether each successive band occurs in the A, C, G or T lane.

Reading the bands upward provides the sequence of the newly synthesized DNA strand in the 5′ → 3′ direction.

If the original template strand is required, its complementary sequence and orientation must also be considered.

Successful sequencing depends on the complete molecular biology workflow and not only on the sequencing instrument.

Sanger sequencing commonly provides usable sequence information across several hundred nucleotides from a sequencing primer.

Under optimized conditions, useful sequence may approach approximately one kilobase, although the usable read length is not a fixed value.

It can depend on:

• DNA-template quality
• primer performance
• DNA sequence composition
• sequencing chemistry
• electrophoretic resolution
• instrument performance
• sequence-quality criteria

One of the most common uses of Sanger sequencing in molecular biology is verification of recombinant plasmids.

A typical cloning-to-sequencing workflow may include:

Colony PCR or restriction analysis can provide evidence that a plasmid contains an insert of the expected approximate size, but these approaches do not directly establish the complete nucleotide sequence.

Sanger sequencing can provide nucleotide-level confirmation of:

• insert identity
• insert orientation
• cloning junctions
• designed mutations
• coding sequences
• selected regulatory elements

A common workflow is:

Specific amplification is important because substantial quantities of unintended PCR products can introduce additional templates and produce overlapping sequence signals.

When sequence accuracy is important, researchers may also consider the fidelity of the DNA polymerase used during upstream PCR amplification.

Sanger sequencing and next-generation sequencing both determine nucleotide sequences, but they are optimized for very different experimental scales.