With the rapid development of science and technology, gene sequencing technology has experienced a huge leap from the first generation to the second generation. Next-Generation Sequencing (NGS) is a revolutionary technology that can quickly and accurately determine a large number of DNA sequences in a short time. It greatly improves the efficiency and accuracy of sequencing, and also greatly reduces the cost of sequencing, making large-scale genome sequencing possible.

What is the principle of second-generation gene sequencing

1. Sequencing while synthesis During the DNA replication process, as the DNA polymerase moves along the template strand, new bases are continuously added to the newly synthesized DNA strand. It is during this process that next-generation gene sequencing technology captures the newly added base information to determine the sequence of DNA. Specifically, during the sequencing process, DNA polymerase, adapter primers, and four kinds of dNTPs (deoxyribonucleoside triphosphates) with base-specific fluorescent labels are added simultaneously. The 3'-OH ends of these dNTPs are chemically protected so that only one dNTP can be added at a time. When a dNTP is added to the synthetic chain, all unused free dNTPs and DNA polymerase are washed away, and then the buffer required to excite fluorescence is added to excite the fluorescence signal. Finally, computer analysis is used to convert the optical signal into sequencing bases.

2. Reversible termination of terminal gene second-generation sequencing technology uses a special reversible terminator. A reversibly terminated end is formed when DNA polymerase incorporates it into a newly synthesized DNA strand. This termination end temporarily prevents the DNA polymerase from adding the next base, allowing only one base to be added at a time and the fluorescent signal captured. Subsequently, by adding specific chemical reagents, this reversible termination end can be cut off, exposing the 3'-OH end for the addition of the next base and sequencing.

3. Sequencing platform and principle example Take the Illumina sequencing platform as an example. Its sequencing principle is based on sequencing-by-synthesis technology. During the sequencing process, DNA fragments are connected to the flow cell through adapters, and clusters are formed after PCR amplification. Then, by adding fluorescently labeled reversible terminal terminator bases in rounds, one base at a time and detecting the emitted fluorescent signal, new strands are gradually synthesized to read the DNA sequence. Specifically: 1. DNA library construction: Cut the DNA sample into short fragments, and add adapters for modification to facilitate subsequent sequencing reactions. 2. On-machine sequencing: Load the constructed DNA library onto the sequencer and perform bridge PCR amplification to form a cluster structure and enhance the fluorescence signal intensity. 3. Sequencing while synthesis: During the sequencing process, fluorescently labeled reversible terminal terminator bases are added in rounds. After each base is added, laser scanning is used to capture the fluorescence signal and determine the type of base added. Then, the reversibly terminated end is excised and the next base is added and sequenced.

4. Technical Characteristics and Applications Gene second-generation sequencing technology has the advantages of high throughput, high efficiency, and low cost. It can sequence a large number of DNA molecules at the same time and is suitable for genomics research, transcriptomics research, disease diagnosis and treatment, etc. fields. For example, in genomics research, second-generation sequencing technology can be used to quickly obtain genomic information of a large number of individuals, revealing key information such as human genetic diversity and disease susceptibility genes; in disease diagnosis, gene mutations in patient samples can be detected , integrating genetic and other information to provide strong support for precision medicine.

The principle of next-generation gene sequencing is mainly based on sequencing-by-synthesis and reversible termination technology, which determines the sequence of DNA by capturing newly added base information. This technology has many advantages and broad application prospects, and plays an important role in life science research and clinical applications.

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