Nucleic acid sequencing method based on fluorescence quenching

Abstract

A nucleic acid sequencing method based on fluorescence quenching is: 1) preparing a sequencing template: the nucleic acid fragments to be sequenced, where the nucleic acid fragments include deoxyribonucleic acid, ribonucleic acid or their nucleic acid sequence amplification products, cloned products, and The product is immobilized on the carrier, 2) On-chip extension: Extend with reaction buffer, polymerase and fluorescently labeled dNTP mixture, 3) Fluorescence quenching: Use a fluorescent quencher to quench the fluorescence, 4) On-chip Extension: use reaction buffer, polymerase and fluorescently labeled dNTP mixture to extend, 5) Image recognition: use Matlab to extract feature points from the obtained extended image, remove noise points to achieve chip analysis or other commercial The optimized software directly processes the image. The invention combines nucleic acid microarray chip technology, and can perform large-scale sequencing of nucleic acids with high throughput, rapidity and low cost.

Description

technical field [0001] The invention is a nucleic acid sequencing method based on fluorescence quenching, which belongs to the genome sequencing method in the field of biomedicine. Background technique [0002] At present, it takes about tens of millions of dollars and half a year to complete the sequencing of a mammalian genome. The cost of DNA sequencing is decreasing by half every two years, but the current general-purpose DNA sequencing methods are still costly and time-consuming, far from meeting the requirements of life science and medical development, and greatly restricting the DNA sequencing market. development of. People urgently need to develop rapid and cheap individualized gene 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 immobilizing the rollin...

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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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