Nucleic acid sequencing method based on fluorescence quenching

Abstract

The invention relates to a nucleic acid sequence detecting method based on fluorescence quenching. The method includes the steps of: firstly, sequence detecting template preparation: the product of anucleic acid fragment to be detected comprises deoxyribonucleic acid, ribonucleic acid or a nucleic acid sequence enlarged product, the cloned product comprises deoxyribonucleic acid and ribonucleic acid are fixed on carrying agent, secondly, extension in the carrying agent: the mixed fluid of reaction buffer fluid, polymerase and fluorescence marked dNTP are used to perform extension, thirdly, fluorescence quenching: fluorescence quenching agent is used to perform fluorescence quenching, fourthly, extension in the carrying agent: the mixed fluid of the reaction buffer fluid, the polymerase and the fluorescence marked dNTP are used to perform extension, fifthly, image recognition: characteristic point extraction is performed to the obtained extended images, the noise point is eliminated torealize analysis to the chip, or other commercialized software is directly used to perform image processing. The invention combines the nucleic acid micro array chip technology, and can perform large-scale nucleic acid sequencing quickly in high flux and at low cost.

Description

Technical field [0001] The invention is a nucleic acid sequencing method based on fluorescence quenching, and belongs to a genome sequencing method in the field of biomedicine. Background technique [0002] At present, it takes tens of millions of dollars and half a year of work to complete the sequencing of the entire mammalian genome. The cost of DNA sequencing is decreasing by half every two years, but the current general DNA sequencing methods are still costly and time-consuming, far from meeting the requirements of life science and medical development, and also greatly restricting the DNA sequencing market development of. People urgently need to develop fast and cheap individualized genetic information detection technology. [0003] Lu Zuhong et al. proposed to prepare a high-density single-molecule multi-copy random whole-genome DNA sequencing template array for the whole genome through single-molecule multi-copy whole-genome amplification. [1] , By fixing the rolling circl...

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Problems solved by technology

[0003] Lu Zuhong et al proposed to prepare a high-density single-molecule multi-copy random whole-genome DNA sequencing template array for the whole genome through single-molecule multi-copy whole-genome amplification [1] , by immobilizing the rolling circle product on the carrier, on the basis of which, on-sheet extension sequencing is performed, each extension of a base has a certain fluorescence intensity, when the fluorescence intensity accumulates to a certain level, it is necessary to quench the fluorescein before further extension, and It is most suitable when no light is needed. There are foreign literatures that use laser to irradiate diphenyl iodine chloride (DPI) to quench fluorescence and then extend [2] , but the damage to DNA is relatively large, DPI drugs are expensive, toxic, and the way of light has certain limitations on high-throughput gene chip sequencing

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