DNA sequencing technology methods and processes

DNA sequencing technology is an important tool in modern biological research, which can accurately determine the nucleotide sequence of DNA in organisms. Through this technology, scientists can gain an in-depth understanding of genome structure, function and variation, providing important data support for fields such as medicine, agriculture and environmental science.

DNA sequencing technology

1. Basic Principle The basic principle of DNA sequencing technology is to use a DNA polymerase to extend the primer bound to the template of the sequence to be determined until a chain-terminating nucleotide is incorporated. Each sequence assay consists of a set of four separate reactions, each containing all four deoxynucleoside triphosphates (dNTPs) mixed with a limited amount of a different dideoxynucleoside triphosphate (ddNTP). Since ddNTP lacks the 3-OH group required for extension, the extended oligonucleotide selectively terminates at G, A, T, or C. The termination point is determined by the corresponding dideoxygen in the reaction, thereby obtaining the DNA sequence.

2. Main methods 1. Sanger dideoxy chain end termination method: This is one of the earliest and most widely used sequencing methods. This method uses DNA polymerase to terminate the chain synthesis if it encounters dideoxynucleotides (ddNTPs) when synthesizing the DNA chain. Four different ddNTPs are terminated at four bases: A, T, C, and G, forming a series of DNA fragments of different lengths. After these fragments are separated by electrophoresis, the DNA sequence can be read. 2. Chemical degradation method: This method was invented by Maxam and Gilbert. It has different principles from the Sanger method, but it can also achieve DNA sequencing. It uses specific chemical reagents to break the DNA chain at specific bases to form a series of DNA fragments of different lengths. After these fragments are also separated by electrophoresis, the DNA sequence can be read. 3. Next-generation sequencing technology: including high-throughput sequencing, single-molecule sequencing and other technologies. These technologies have the advantages of fast sequencing speed, low cost, and high accuracy. They use advanced sequencing platforms and algorithms to sequence a large number of DNA fragments at the same time, greatly improving sequencing efficiency.

3. Sequencing process 1. The general process of DNA sequencing technology includes DNA extraction, DNA fragmentation, DNA library construction, DNA amplification, sequencing and sequencing result analysis. 2. DNA extraction: DNA is isolated and purified from cells or tissues through biochemical methods. 3. DNA fragmentation: Break long-chain DNA into short fragments for subsequent sequencing reactions. 4. DNA library construction: The fragmented DNA is specially processed to construct a sequencing library for high-throughput sequencing. 5. DNA amplification: Amplify DNA through technologies such as PCR to increase the amount of DNA for sequencing. 6. Sequencing: Use a sequencing instrument to perform a sequencing reaction on the amplified DNA to obtain original sequencing data. 7. Sequencing result analysis: Use bioinformatics methods to process and analyze the sequencing data to obtain the final DNA sequence information.

4. Application fields DNA sequencing technology has extensive application value in many fields: 1. Basic scientific research: used to study the structure and function of genes, the composition and evolution of genomes, etc. 2. Medical field: used for diagnosis of genetic diseases, formulation of personalized medical plans, drug research and development, etc. 3. Forensic science: used for evidence analysis and identification at crime scenes, etc. 4. Agricultural field: used for the improvement of crop varieties, protection of genetic diversity, etc.

With the continuous development of technology, DNA sequencing technology is developing in the direction of higher throughput, lower cost, and higher accuracy. At the same time, sequencing data processing and analysis methods are also constantly improving, providing strong support for the widespread application of DNA sequencing technology. In the future, DNA sequencing technology is expected to play an important role in more fields and make greater contributions to the development of human society.

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