The article provides a comprehensive overview of the evolution of DNA sequencing technology over the past fifty years. It begins by highlighting the importance of nucleic acid sequencing in biological research, emphasizing that understanding the sequence of nucleic acids is crucial for comprehending the hereditary and biochemical properties of life forms. The first-generation sequencing techniques, such as those developed by Robert Holley, Fred Sanger, and others, focused on sequencing relatively short RNA molecules and used methods like ribonuclease digestion and two-dimensional fractionation. These techniques gradually progressed to DNA sequencing, with the development of the Sanger 'chain-termination' method, which used dideoxy nucleotides to terminate DNA polymerization and infer the sequence by electrophoresis. The second-generation sequencing technologies, including pyrosequencing and high-throughput methods like those from 454 Life Sciences and Illumina, revolutionized the field by enabling parallel sequencing of multiple DNA molecules. These techniques used luminescent methods to measure pyrophosphate synthesis and fluorescent dyes to monitor nucleotide incorporation, significantly increasing the speed and efficiency of sequencing. The third-generation sequencing technologies, such as single molecule real-time (SMRT) sequencing and nanopore sequencing, further advanced the field by allowing single-molecule sequencing without the need for amplification. These technologies offer longer read lengths and higher accuracy, with applications in de novo genome assemblies and real-time, decentralized sequencing. The article concludes by emphasizing the importance of DNA sequencing in biological research and the rich history of its development, highlighting how innovations in sequencing protocols, molecular biology, and automation have transformed the field.The article provides a comprehensive overview of the evolution of DNA sequencing technology over the past fifty years. It begins by highlighting the importance of nucleic acid sequencing in biological research, emphasizing that understanding the sequence of nucleic acids is crucial for comprehending the hereditary and biochemical properties of life forms. The first-generation sequencing techniques, such as those developed by Robert Holley, Fred Sanger, and others, focused on sequencing relatively short RNA molecules and used methods like ribonuclease digestion and two-dimensional fractionation. These techniques gradually progressed to DNA sequencing, with the development of the Sanger 'chain-termination' method, which used dideoxy nucleotides to terminate DNA polymerization and infer the sequence by electrophoresis. The second-generation sequencing technologies, including pyrosequencing and high-throughput methods like those from 454 Life Sciences and Illumina, revolutionized the field by enabling parallel sequencing of multiple DNA molecules. These techniques used luminescent methods to measure pyrophosphate synthesis and fluorescent dyes to monitor nucleotide incorporation, significantly increasing the speed and efficiency of sequencing. The third-generation sequencing technologies, such as single molecule real-time (SMRT) sequencing and nanopore sequencing, further advanced the field by allowing single-molecule sequencing without the need for amplification. These technologies offer longer read lengths and higher accuracy, with applications in de novo genome assemblies and real-time, decentralized sequencing. The article concludes by emphasizing the importance of DNA sequencing in biological research and the rich history of its development, highlighting how innovations in sequencing protocols, molecular biology, and automation have transformed the field.