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Gene Sequencing
Gene sequencing technology, as a core tool in modern life sciences, has progressed from laboratory research to clinical diagnosis and personalized medicine, profoundly transforming our understanding of life's essence and approaches to disease diagnosis and treatment.
1. Basic Principles of Gene Sequencing Technology
The core objective of gene sequencing technology is to determine the sequence of the four bases (A, T, C, G) in a DNA molecule, which carries all the genetic instructions of an organism. Since Frederick Sanger invented the chain termination method (first-generation sequencing) in 1977, gene sequencing technology has undergone three revolutionary leaps, each time leading to exponential growth in sequencing throughput and a significant decrease in cost.
Principle of First-Generation Sequencing (Sanger Method)
The dideoxy chain-termination method, representing first-generation sequencing technology, is based on the use of 2',3'-dideoxynucleotides (ddNTPs) as chain terminators during DNA strand elongation.
Principle of Second-Generation Sequencing (NGS)
The revolutionary breakthrough of second-generation sequencing lies in immobilizing DNA fragments on a solid surface and achieving ultra-high throughput sequencing through massively parallel reactions.
Principle of Third-Generation Sequencing
The essential characteristic of third-generation sequencing is real-time single-molecule sequencing, which eliminates the need for PCR amplification and allows direct detection of native DNA molecules.
2. Key Performance Metrics of Sequencing Technologies
The development of gene sequencing technology has been driven primarily by the demands of the Human Genome Project and large-scale population genomics studies. As the technology moved from large research centers to ordinary laboratories and clinical institutions, it gave rise to emerging fields such as cancer genomics, microbiomics, and single-cell sequencing.
Key performance metrics for sequencing technologies include:
Read Length: The number of consecutive bases obtained in a single sequencing run. Longer read lengths facilitate genome assembly and structural variation detection.
Accuracy: The probability of correct base calling. Clinical applications typically require >99.9%.
Throughput: The amount of data generated in a single run, which determines project scale and cost.
Depth: The average number of times a genomic region is covered, affecting variant detection sensitivity.
Different application scenarios prioritize these metrics differently. For example, screening for tumor mutations requires high accuracy and moderate depth (30-100X), while novel genome assembly requires long read lengths and high throughput.
With the maturation of sequencing technology, multi-omics integration and real-time analysis have become new trends. Correlating DNA sequencing data with transcriptomic (RNA-seq), epigenomic (ChIP-seq), and proteomic (mass spectrometry) data enables a comprehensive analysis of genotype-phenotype relationships. Real-time sequencing technologies, like Oxford Nanopore, support immediate analysis during data generation, showing unique advantages in rapid infectious disease diagnosis and field-based biological monitoring. In the future, fourth-generation technologies such as quantum sequencing and electron microscopy sequencing may further break existing limitations, enabling faster and more economical genome decoding.
3. Workflow of DNA Sequencing
Despite differences in technical principles, modern sequencing experiments generally follow a similar standard workflow:
3.1 Sample Preparation: Extract high-quality DNA/RNA from samples such as blood or tissue, performing whole-genome amplification if necessary.
3.2 Library Construction: Fragment DNA (via ultrasonication or enzymatic digestion), perform end repair, and ligate sequencing adapters. This step may include size selection and PCR enrichment. Targeted sequencing also requires hybrid capture or amplicon PCR.
3.3 Cluster Generation/Template Preparation: Second-generation sequencing typically requires in vitro amplification to generate sufficient signal, such as bridge PCR (Illumina) or emulsion PCR (Ion Torrent). Third-generation sequencing can directly use native DNA molecules.
3.4 Sequencing Reaction: Run the corresponding chemical reactions based on the platform's characteristics, ranging from several hours (Ion Torrent) to several days (HiSeq X).
3.5 Data Analysis: Convert raw images or signals into base sequences (base calling), followed by quality filtering, alignment to a reference genome, variant detection, and other downstream analyses.
4. Application Fields of Gene Sequencing Technology
By analyzing the base sequences of DNA or RNA, gene sequencing technology has permeated all areas of life sciences and medicine. Its broad and deep applications, from basic research to clinical use, and from human health to agricultural breeding, are continuously driving revolutionary progress in related fields.
Typical application areas include:
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Agricultural Breeding Research | Human Genetic Research |
5. Typical CNI Light Sources for Gene Sequencing
Currently, most DNA sequencing methods rely on laser-induced fluorescence technology, using different fluorescent dyes to represent the four different nucleotides (A, C, G, T). Typical requirements involve two-wavelength and four-wavelength combined light sources. CNI offers a rich selection of wavelengths matched to the absorption peaks of each fluorescent marker. Typical wavelengths include 320nm, 335nm, 349nm, 355nm, 365nm, 375nm, 405nm, 430nm, 450nm, 473nm, 488nm, 515nm, 532nm, 561nm, 577nm, 589nm, 633nm, 639nm, 670nm, 690nm, etc., with power levels ranging from hundreds of milliwatts to several watts.
Additionally, gene sequencing systems often use multi-wavelength LED combined light sources. Typical wavelengths are 365nm, 385nm, 405nm, 440nm, 460nm, 470nm, 480nm, 525nm, 527nm, 555nm, 579nm, 590nm, 630nm, 650nm, 730nm, and white light sources. For detailed specifications, please visit www.cnilaser.com.
A complete gene sequencing run typically takes tens of hours, placing very strict requirements on the long-term wavelength and power stability of the laser. Generally, the RMS stability over 100 hours must be better than 1%, alongside requirements for spectral purity and laser band blocking with an OD value greater than 5. Custom uniform light field output is available with a uniformity better than 90%.
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Superior to 90% of light fields Homogenization Technology |
Changchun New Industries Optoelectronics Technology Co., Ltd.
Address: No. 888 Jinhu Road, High-Tech Zone, Changchun, 130103, P.R.China
International Tel:+86-431-85603799
Email: sales@cnilaser.com
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