Automated Laser Fluorescence DNA Sequencing

Summary

At present, the application of automated laser fluorescence DNA sequencing technology (also known as first generation sequencing technology) has been very common. The basic principles of automated laser fluorescence DNA sequencing are all based on Sanger double deoxygenation, but the specific working principles of different automated laser fluorescence DNA sequencing systems are not the same, in general, the working principle can be divided into two categories of four-color fluorescence and single-color fluorescence method.

Principle

The basic principles of automated laser fluorescence DNA sequencing are all based on the Sanger double deoxygenation method, but the specific working principles of different automated laser fluorescence DNA sequencing systems are different, and in general, the working principles can be divided into two categories: four-color fluorescence method and monochromatic fluorescence method.

(I) Four-color fluorescence method

Using four different fluorescent dyes to label the same primer or four different termination substrate ddNTP, the final result is equivalent to give the DNA fragment four different colors, therefore, a sample of four reaction products can be electrophoresed in the same lane, thus reducing the different sequencing lanes between the difference in electrophoretic mobility of the sequencing results of the impact on the accuracy of the sequencing results. There are two specific methods:

1. 4 fluorescent dyes are labeled at the 5'-end of the same sequencing primer, thus forming a group of (4) labeled primers. In a Sanger sequencing reaction, a specific fluorescently labeled primer corresponds to a specific rlrlNTP substrate, so that all DNA fragments terminated with a certain drlNTP are labeled with the same fluorescent group at the 5'-end.

2. 4 fluorescent dyes are labeled on each of the 4 drlNTP substrates, and after the Sanger reaction, the 3'-ends of the reaction products are labeled with different fluorescence.

(ii) Monochromatic fluorescence method

A single fluorescent dye is used to label the 5'-end of the primer or dNTP, and after the Sanger reaction, the 5'-ends of all the products are labeled with the same fluorescence (i.e., equivalent to giving the same color to all the DNA fragments), so the four reactions of a sample must be carried out separately, and the corresponding products must also be electrophoresed in four different lanes.

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