DNA sequencing is a fundamental tool in modern biology, allowing scientists to determine the precise order of nucleotides in a DNA molecule. This information is crucial for understanding genetic variations, identifying pathogens, and studying gene function. Among the various methods developed, Sanger sequencing stands out as a foundational technique. Despite the advent of newer, high-throughput technologies, Sanger sequencing continues to hold relevance in scientific research today.
Sanger Sequencing Explained
Sanger sequencing, often called the chain termination method, operates by selectively stopping DNA strand elongation. This process involves using a DNA template, a primer, DNA polymerase, and a mixture of normal deoxynucleotide triphosphates (dNTPs) along with modified dideoxynucleotide triphosphates (ddNTPs). The ddNTPs lack a crucial chemical group needed for further DNA chain extension. Each of the four ddNTPs (ddATP, ddGTP, ddCTP, ddTTP) is labeled with a distinct fluorescent dye.
During the sequencing reaction, DNA polymerase incorporates dNTPs to build a new strand complementary to the template. When a fluorescently labeled ddNTP is randomly incorporated, it terminates the growing DNA chain, creating DNA fragments of various lengths. These fragments are then separated by size using capillary electrophoresis, with shorter fragments moving faster. A laser excites the fluorescent dyes at the end of each fragment, and a detector records the color, allowing the sequence of the original DNA template to be determined by the order of the fluorescent signals.
Where Sanger Sequencing Excels Today
Sanger sequencing remains valuable in several specific applications due to its accuracy and ability to produce long, high-quality reads for targeted DNA regions. One primary use is the validation of results obtained from Next-Generation Sequencing (NGS) studies. Because Sanger sequencing offers high accuracy, around 99.99%, it is considered a reliable method for confirming genetic variants identified by high-throughput NGS, especially for clinical decision-making.
For analyzing single genes or small, targeted regions of DNA, Sanger sequencing is often the preferred method. It is cost-effective and efficient for projects involving a limited number of samples or genetic targets. This includes applications like single nucleotide polymorphism (SNP) detection, where precise identification of a specific base change is required.
Sanger sequencing plays a role in bacterial identification, particularly by sequencing the 16S ribosomal RNA gene. This method is effective for identifying atypical bacteria that are challenging to culture and can provide species-level identification for many samples. It is also routinely used for verifying cloning experiments and plasmid sequences in molecular biology laboratories. This ensures inserted DNA fragments are correct.
Sanger’s Role Alongside Next-Generation Methods
The development of Next-Generation Sequencing (NGS) technologies revolutionized DNA sequencing by offering higher throughput and lower costs for large-scale projects. NGS can sequence millions of DNA fragments simultaneously, making it suitable for whole-genome sequencing, transcriptomics, and novel variant discovery. This contrasts with Sanger sequencing, which processes a single DNA fragment at a time, limiting its scalability for extensive genomic analyses.
Despite the capabilities of NGS, Sanger sequencing is not obsolete; instead, it serves a complementary role. NGS excels in broad discovery, identifying genetic variations across large regions, but may have limitations in detecting low-frequency variants or in regions with complex genomic structures. In such cases, Sanger sequencing provides the precision to confirm specific variants or fill gaps in NGS data, especially in difficult-to-sequence areas like those with high GC content or repetitive elements.
Its established workflow and ability to produce long, accurate reads (500-1000 base pairs) make it a reliable tool for targeted analysis, as sequencing technologies evolve. Therefore, both Sanger and NGS technologies offer distinct advantages and are chosen based on the specific research question and required scale.