DNA sequencing can be divided into manual sequencing and automatic sequencing. Manual sequencing includes Sanger dideoxy chain termination method and Maxam-Gilbert chemical degradation methods. Automated sequencing has actually become the mainstream of DNA sequence analysis. Automatic sequencing is developed on the basis of Sanger method.
The principle of p>DNA sequencing should be based on the principles of Maxam-Gilbert chemical degradation, Sanger dideoxy chain termination method and now developed pyrophosphorylated sequencing.
If the most commonly used sequencing principle is automatic sequencing: capillary electrophoresis technology is used to replace the traditional polyacrylamide plate electrophoresis, and ddNTP (labeled terminator method) with four-color fluorescent dyes patented by the company is applied, so the PCR products generated by single primer PCR sequencing reaction are single-stranded DNA mixtures with four different fluorescent dyes at the 3' end with a difference of one base, so that the sequencing PCR products of four fluorescent dyes can be electrophoresed in one capillary tube, thus avoiding swimming lanes. Due to the different sizes of molecules, the mobility in capillary electrophoresis is different. When the molecules pass through the capillary reading window, the CCD(charge-coupled device) camera detector in the laser detector window can detect the fluorescent molecules one by one. The excited fluorescence is split by grating to distinguish the fluorescence of different colors representing different base information, and the images are synchronized on the CCD camera. The analysis software can automatically convert different fluorescence into DNA sequences, thus achieving the purpose of DNA sequencing.
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The principle of genome sequencing involves sample preparation, large-scale sequencing and sequence analysis
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