Illumine
Illumina's Solexa and Hiseq should be the most famous and widely-used machines for the next generation sequencing in the world. The core principle of these two technical series are the same, which is sequencing at the same time of gene synthesis. The process is divided into the following 4 steps.
A.DNA Library Construction
Use ultrasonic wave to break DNA samples into small fragments of 200-500bp, and add different adapters at both ends of these small fragments which finally form the single stranded DNA library.
B. Flow Cell
Flow cell is a channel for adsorption of flow DNA fragments. When the library is built, the DNA in these libraries will be randomly attached to the channel of flow cell surface when flowing through. Each flow cell has 8 channels. The surface of each channel has a lot of adapters. These adapters will make pairs with those at both ends of DNA segments in the process of library building, and can support the DNA amplification by bridge PCR.
C. Bridge PCR amplification and denaturation
Bridge PCR takes adapters fixed on flow cell surface as templates. After amplification and denaturation cycles, each DNA fragment will bunch with many copies in their respective positions. Each bundle contains a single DNA template. This process aims to achieve the amplification fluorescent signal of bases, so as to achieve required signal for sequencing.
D. Sequencing
The core method is that sequencing at the same time of gene synthesis. DNA polymerase, adapter primers and dNTPs with base specific fluorescent labeling (like Sanger sequencing) are added to the reaction system simultaneously. These 3 '-OH groups of dNTPs are protected by chemical methods, so only one dNTP can be added at a time. After dNTP is added to the synthetic chain, all unused free dNTPs and DNA polymerases are washed off. Then, add the needed buffer for triggering fluorescence with the laser, and record the fluorescence signal with optical equipment, which would be analyzed to get sequence result by computer software. In the end, a kind of chemical reagent is added to quench the fluorescent signal and remove protection groups of dNTPs 3 '-OH, so as to carry out the next run of sequencing reaction.
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