A two-nucleotide real-time sequencing-by-synthesis method with reversible 3' end blocking

Abstract

The invention discloses a 3'end reversible closed two-nucleotide real-time synthesizing and sequencing method. A single sequencing reaction is performed on two different 3-end modified nucleotides of X, Y simultaneously; a whole sequencing method comprises: performing at least two groups of sequencing reactions on the same template: each group of the sequencing reaction involving four 3-end modified nucleotides is to perform a circulation of synthesizing and sequencing reactions by utilizing two different nucleotides simultaneously according to a manner that each nucleotide is only used once in one circulation, and after a plurality of sequencing reactions are completed, a group including a plurality of XY information arranged according to a sequencing order is obtained; after the group of sequencing reaction is completed, denaturating to clear away an extension chain of a sequencing primer, repeatedly hybridizing the sequencing primer, and performing a second group of sequencing reaction, thereby obtaining a plurality of XY information arranged according to the second sequencing reaction; and finally assembling a specific base sequence of a to-be-tested nucleic acid segment by decoding the two groups including the plurality of XY information arranged according to the sequencing orders. The 3'end reversible closed two-nucleotide real-time synthesizing and sequencing method provided by the invention can eliminate sequencing errors of the existing real-time synthesizing and sequencing method to a homopolymer segment.

Description

technical field [0001] The invention belongs to the field of biotechnology, and is a method for realizing high-throughput determination of nucleic acid sequences, in particular to a two-nucleotide real-time synthetic DNA sequencing method and its application. Background technique [0002] With the development and completion of the Human Genome Project and various model organism genome projects, human beings have entered the post-gene era, which has had a huge impact on contemporary biological research and medical research, and the related disciplines of molecular biology have been rapidly developed. develop. It will become possible to understand the differences of life, the law of disease occurrence and development, and the interaction between drugs and living organisms at the genetic level. Drastically reducing the cost of DNA sequencing will greatly promote research in life sciences and medicine, and even bring about revolutionary changes. At present, whole-genome DNA se...

AI Technical Summary

Problems solved by technology

However, this patented technology still has some shortcomings. For homopolymers (such as fragments such as AAAAAAAAAA) or quasi-homopolymers (such as fragments such as AACCAAACAACAA in AC / GT cycle sequencing), the signal intensity and base number obtained by sequencing The number does not form a complete linear relationship, resulting in errors in sequencing information

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