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Circular 'connection-extension' genome sequencing method

A genome sequencing and ligation technology, applied in the field of biomedicine, can solve the problems of high sequencing cost, difficulty in resequencing the human whole genome with high coverage, etc., and achieve the effect of great application prospects.

Inactive Publication Date: 2010-01-27
SOUTHEAST UNIV
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Problems solved by technology

Since current DNA sequencing methods use PCR products as sequencing templates, it is difficult to achieve high-coverage resequencing of the human genome, and the cost of sequencing is still high

Method used

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  • Circular 'connection-extension' genome sequencing method
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  • Circular 'connection-extension' genome sequencing method

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Embodiment Construction

[0055] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0056] see figure 1 , the genome is composed of four bases: A, T, C, and G. Combining the four bases in pairs, we artificially define the following four-color definition matrix, so that we can combine all of a sequence Adjacent bases are defined by colors, and fluorescently labeled probes of corresponding colors are used for sequencing.

[0057] see figure 2 , the length of the specific probe is eight bases, and the fourth and fifth bases are specific binding sites. When the bases at these two positions are complementary to the bases at the corresponding positions of the template strand, A fluorophore attached to the probe tail then emits light.

[0058] see image 3 , is the sequencing step (1) the universal primer is connected to the initial fragment of the template strand, the probe mixture reacts with the template, and the fourth and fifth pro...

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Abstract

The invention relates to a circular 'connection-extension' DNA sequencing method. The method comprises the following steps: based on the sequencing principle of an SOLiD sequencing technology, adopting a sequencing primer to connect a section of universal oligonucleotide, detecting the base information of a corresponding position of a DNA template by extending a base, then cutting a marking object from a certain position in the oligonucleotide connected with the sequencing primer, and according the same method, extending one marking monomer after reconnecting the same oligonucleotide, and then sequentially determining the base information of a plurality of spacing positions on the DNA template; eliminating the sequencing primer finishing the determination of the base sequences of a plurality of spacing positions by a denaturation method or a digestion method, changing a new sequencing primer, and determining the base information of a plurality of other spacing positions on the DNA template by the 'connection-extension' method in the same way; circularly changing sequencing primers, and finally combining the base information of the spacing positions determined by these sequencing primers to obtain arrangement information of all bases of certain fragment of the DNA template so as to realize the DNA sequence detection.

Description

technical field [0001] The invention relates to gene sequencing technology, in particular to a cycle "connection-extension" genome sequencing method, which belongs to the technical field of biomedicine. Background technique [0002] Existing gene sequencing technologies include the following [0003] 1. Pyrosequencing [0004] Pyrosequencing (Pyrosequencing) technology is a new generation of DNA sequence analysis technology, this technology does not need electrophoresis, DNA fragments do not need fluorescent labeling, and the operation is extremely simple. Pyrosequencing technology is an enzyme cascade chemiluminescent reaction in the same reaction system catalyzed by four enzymes. In each round of sequencing reaction, only one dNTP is added. If the dNTP is paired with the template, the polymerase can incorporate it into the primer strand and release an equimolar number of pyrophosphate groups (PPi). PPi can be finally converted into a visible light signal and converted i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 陆祖宏薛易旻孙峰金露潘旻
Owner SOUTHEAST UNIV
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