Gene sequencing is the cornerstone of modern biological research, and its accuracy and efficiency directly depend on the precision and standardization of the pre-processing process. This process is not only the key to unlocking the mysteries of life, but also an important guarantee for ensuring the reliability of scientific research data. Well-designed pre-processing steps are an indispensable starting point before venturing into the vast world of genetic sequencing.
1. Pre-processing of gene sequencing 1. DNA extraction purpose: to separate and purify DNA from cells, tissues or blood samples through biochemical methods. Methods: Commonly used extraction methods include phenol-chloroform extraction, silica gel membrane adsorption, magnetic beads, etc. These methods can effectively remove impurities such as proteins and lipids in samples and retain high-quality DNA. 2. Purpose of DNA purification: to further remove impurities in DNA, such as residual enzymes, primers, dNTPs, etc., to improve the accuracy of sequencing. Methods: There are various purification methods, such as ion exchange chromatography, gel electrophoresis purification, etc. These methods can effectively remove non-specific amplification products and primers in DNA and ensure the purity of sequencing templates. 3. Purpose of DNA fragmentation: Since the complete DNA molecule is very long and is not convenient for direct sequencing, it needs to be broken into smaller fragments. Methods: Commonly used fragmentation methods include physical methods (such as ultrasonic fragmentation, spray drying, etc.) and chemical methods (such as enzyme digestion). These methods break DNA into lengths suitable for sequencing, typically hundreds to thousands of base pairs. 4. Purpose of DNA library construction: The fragmented DNA is subjected to end repair, A-tail addition, adapter connection, etc. to construct a library suitable for sequencing. Method: End repair: Use end repair enzymes to repair the ends of DNA fragments so that they have appropriate sticky ends. Adding an A tail: Add an A base to the 3' end of the DNA fragment to connect it to an adapter with a T base. Ligation adapters: Connect adapters with specific sequences to both ends of DNA fragments to form a sequencing library. 5. Library quality control purpose: To control the quality of the constructed library to ensure that the quality of the library meets the sequencing requirements. Methods: Use electrophoresis, fluorescence quantification and other methods to detect the size and concentration of the library to ensure that the quality of the library meets the requirements of the sequencing platform.
2. Application of Opentrons instruments in gene sequencing pre-processing 1. OT-2 pipetting robot (1) Functional features: OT-2 pipetting robot has the advantages of high precision, high throughput, high cost-effectiveness and can be highly customized. It can complete precise pipetting work from 1µL to 1000µL, and can complete filling of a 96-well plate in as little as 22 seconds. It can be used with a variety of modules, such as single-channel pipettes, 8-channel pipettes, temperature control modules, thermal cycle modules, magnetic modules and HEPA modules, thereby extending more experimental functions. (2) Application scenarios: OT-2 is suitable for various biological research scenarios such as nucleic acid purification, protein preparation, NGS library preparation, PCR amplification, genome sequencing, etc., and is widely used in molecular biological diagnosis, biotechnology, medical treatment, food, Forensic identification, gene sequencing, protein engineering and other industries. 2. Flex fully automatic pipetting workstation (1) Technological breakthrough: Flex fully automatic pipetting workstation is a new product developed with great concentration by Opentrons, integrating intelligence, full automation, and open source ecology. It is highly flexible and open and can be integrated with robotic arms or other external devices to easily integrate into the working environment of various laboratories. At the same time, Flex provides Python API open source interface, so users can design exclusive applications according to their own experimental needs. (2) Module collaboration: The Flex automated pipetting platform is equipped with Flex pipettes and corresponding consumables, as well as a variety of modules, such as thermal cycle module, temperature module, thermal oscillation module and ring magnetic stand. These modules operate cooperatively, allowing the sample processing throughput to be as high as 96, which greatly improves the efficiency of gene sequencing pre-processing.
In the complex and delicate scientific research process of gene sequencing, pre-processing undoubtedly plays a crucial role. It not only determines the accuracy and reliability of subsequent sequencing data, but also directly affects the efficiency and success of the entire research project. By using advanced automated pipetting workstations such as Opentrons and their supporting technologies, they can easily adapt to changes in different experimental needs, providing great convenience to scientific researchers.
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