Cycle sequencing is an automated, highly efficient variant of the classic Sanger sequencing method. This process determines the precise linear order of the four nucleotide bases—adenine (A), guanine (G), cytosine (C), and thymine (T)—within a target DNA segment. By integrating a process similar to the Polymerase Chain Reaction (PCR) with the chain termination chemistry of Sanger sequencing, the technique gained the sensitivity and robustness needed for large-scale genetic projects. This advancement allowed researchers to sequence DNA with much smaller amounts of starting material and significantly higher throughput, revolutionizing the fields of genetics and molecular biology.
The Chemical Foundation of Cycle Sequencing
The core chemistry of cycle sequencing relies on the principle of chain termination, which involves two distinct types of nucleotides. Standard deoxynucleotide triphosphates (dNTPs)—dATP, dGTP, dCTP, and dTTP—are the building blocks used by the DNA polymerase to construct a new DNA strand complementary to the template. These dNTPs possess a hydroxyl group (-OH) at the 3′ position of the deoxyribose sugar, which is necessary for forming the phosphodiester bond that links the next nucleotide in the growing chain.
The reaction mixture also contains dideoxynucleotide triphosphates (ddNTPs). These ddNTPs are the chain terminators, lacking the necessary 3′-OH group found on dNTPs. When a DNA polymerase incorporates a ddNTP into the growing chain instead of a dNTP, the absence of the hydroxyl group means no further nucleotides can be added.
This termination step creates a nested set of DNA fragments. In modern automated sequencing, each of the four ddNTP types is tagged with a unique fluorescent dye, allowing for the simultaneous detection of all four bases in a single reaction tube. The reaction is balanced so that termination occurs randomly at every possible position along the template strand, generating a collection of fragments that vary in length by a single base, each marked by a specific fluorescent color at its final base.
Executing the Sequencing Reaction
The “cycle” component of this technique refers to the repetitive thermal cycling process, a modified version of the protocol used in PCR. This cycling is performed in a thermal cycler to generate a detectable quantity of fluorescently labeled DNA fragments. Unlike standard PCR, which uses two primers to exponentially amplify both strands of a DNA target, cycle sequencing uses only a single primer, resulting in a linear, not exponential, amplification of the terminated fragments.
Each cycle consists of three distinct temperature steps. The first step is denaturation, where the reaction mixture is heated to a high temperature, typically around \(94^\circ\text{C}\) to \(98^\circ\text{C}\), to separate the double-stranded DNA template into single strands. This separation makes the template accessible for the next step.
Following denaturation, the temperature is lowered to an annealing temperature, allowing the short, single-stranded primer to bind to its complementary sequence on the template DNA strand. The primer binding site is immediately upstream of the region intended for sequencing.
Finally, the temperature is raised to the optimal extension temperature for the heat-stable DNA polymerase, often around \(60^\circ\text{C}\) to \(72^\circ\text{C}\). The polymerase begins synthesizing a new strand from the primer, incorporating dNTPs until, purely by chance, a fluorescently labeled ddNTP is incorporated, thereby terminating that specific fragment. By repeating this cycle 25 to 35 times, a sufficient amount of the terminated, labeled DNA fragments is produced for subsequent analysis.
Reading the Results
After the thermal cycling reaction is complete, the mixture contains millions of fluorescently tagged DNA fragments of varying lengths. These fragments must be precisely separated to determine the order of the terminal bases. This separation is accomplished using a technique called capillary electrophoresis (CE).
The fragments are loaded into thin glass capillaries filled with a gel matrix, and an electric current is applied. Because all DNA fragments are negatively charged, they migrate toward the positive electrode. The gel matrix separates the fragments based on size, allowing the smallest fragments to travel fastest and reach the detection point first.
As the fragments pass a specific detection window near the end of the capillary, a laser excites the fluorescent dyes attached to the terminal ddNTPs. A detector records the color of the fluorescence, translating the data into a graph known as a chromatogram or electropherogram, which displays a series of colored peaks corresponding to specific bases (A, T, C, or G). The sequential order of these colored peaks directly reveals the nucleotide sequence of the original DNA template.
Modern Uses in Research and Diagnostics
While newer sequencing technologies have emerged for large-scale genome projects, cycle sequencing coupled with capillary electrophoresis remains the standard for specific applications requiring high accuracy and long read lengths of up to 1000 bases. It is frequently used to verify the sequence of small DNA fragments, such as those created during molecular cloning experiments. For example, researchers use it to confirm the correct integration of a gene into a plasmid or to check the sequence of a small DNA insert.
In clinical diagnostics, cycle sequencing is often preferred for single gene mutation analysis, where high confidence in a specific short sequence is required. This technique is routinely employed for applications like determining the human leukocyte antigen (HLA) type, which is necessary for organ transplantation matching. It also plays a role in forensic science and pathogen identification, as its high accuracy over short reads makes it reliable for confirming the identity of a DNA target.