Monochromatic fluorescence MRT gene sequencing reagent and method based on environment-sensitive dye

Abstract

The invention relates to a monochromatic fluorescence MRT gene sequencing reagent and method based on an environment-sensitive dye, the reagent comprises a polymerase and four different labeled nucleotide analogues, hydroxyl groups at nucleotide 3'positions of the four nucleotide analogues are all modified by a protecting group or a cleavable linking group, the protecting group or the cleavable linking group can be cleaved and separated to expose 3 '-OH again, and the cleavable linking group is linked with the environment high-sensitivity fluorophore or the environment high-sensitivity fluorophore marker through a covalent bond or a non-covalent bond; the four nucleotide analogues are nucleotide derivatives capable of emitting or incapable of emitting fluorescence. The invention also discloses a gene sequencing method by using the reagent. According to the method, polymerization reaction and SBS sequencing can be effectively identified and catalyzed when macromolecular group modification exists at 3 '-O, and the problem of influence on sequencing reading length caused by accumulation of residues on basic groups in a previous second-generation SBS sequencing method is solved.

Description

technical field [0001] The invention relates to the field of gene sequencing, in particular to an environment-sensitive dye-based monochromatic fluorescent MRT gene sequencing reagent and method. Background technique [0002] Sequencing technology is used to determine the primary structure of biological macromolecules such as nucleic acids, proteins, polysaccharides, etc. It is a basic tool for life science research and the main means of obtaining biological information data, especially DNA sequencing technology, which is to obtain the base sequence of the target DNA fragment Obtaining the sequence of the target DNA fragment is the basis for further molecular biology research and genetic modification, enabling researchers to study and better understand health and disease, and is an essential technology for the personalized precision medicine paradigm. According to different principles, the development of sequencing technology can be roughly divided into four historical stage...

AI Technical Summary

Problems solved by technology

The main challenge of this approach is that, in the complex state formed by the polymerase with the complementary nucleotide and the DNA template, the 3'-OH position of the nucleotide is very close to the amino acid residue at the active site of the DNA polymerase. Therefore, it is difficult for DNA polymerase to accept 3'-0 macromolecular dye-modified nucleotides as substrates

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