- First-Generation Sequencing encompasses the earliest set of DNA sequencing technologies developed in the 1970s and 1980s, which provided the first practical means of decoding genetic information. This generation is primarily associated with the Sanger sequencing method, but also includes the lesser-used yet historically significant Maxam–Gilbert sequencing.
- These technologies marked the beginning of genomic science, leading to major achievements such as the sequencing of the first virus genome (φX174) and laying the foundation for the Human Genome Project.
- The most well-known and widely used first-generation method is Sanger sequencing, also called the chain-termination method, developed by Frederick Sanger in 1977. This method involves synthesizing a new DNA strand using a template and incorporating modified nucleotides known as dideoxynucleotides (ddNTPs), which halt DNA synthesis upon incorporation due to the absence of a 3’-hydroxyl group. The resulting DNA fragments, each terminating at a specific base, are separated by length using gel or capillary electrophoresis. Each ddNTP is tagged with a fluorescent dye, allowing for automated detection and accurate base calling. Though labor-intensive and relatively slow, Sanger sequencing produces long and highly accurate reads (typically 500–1000 base pairs), which made it the dominant method for over two decades. It remains a gold standard today for applications requiring precision, such as validating mutations, sequencing plasmids, or performing clinical diagnostics.
- Another important but less widely adopted first-generation technique is Maxam–Gilbert sequencing, developed by Allan Maxam and Walter Gilbert in the same year as Sanger’s method. This technique involves chemical modification and cleavage of DNA at specific bases (adenine, guanine, cytosine, or thymine) followed by electrophoretic separation of the resulting fragments. It does not require a DNA polymerase or prior amplification, which was initially advantageous. However, it uses hazardous chemicals like hydrazine and piperidine, involves radioactive labeling, and requires complex workflows. These drawbacks led to its decline in favor of the safer and more scalable Sanger method.
- First-generation sequencing also benefited from conceptual advances such as the shotgun sequencing strategy, which was essential in large-scale genome projects. In this approach, DNA is randomly fragmented into smaller pieces, each of which is sequenced individually (usually by Sanger sequencing), and then assembled computationally based on overlapping regions. This strategy proved indispensable during the Human Genome Project and demonstrated how first-generation techniques could be scaled with significant effort and computational support.
- Despite their pioneering contributions, first-generation sequencing methods are limited by low throughput, high cost per base, and significant labor and time requirements. These limitations ultimately drove the development of second- and third-generation sequencing technologies, which offer massively parallel processing and higher efficiency. However, first-generation methods, especially Sanger sequencing, continue to be used for small-scale or highly precise applications due to their superior accuracy and reliability.
