A special modified nucleotide and its application in high-throughput sequencing

Abstract

The invention discloses a specially modified nucleotide as well as an application thereof in high-throughput sequencing. A fluorescence marked group of the nucleotide and a nucleotide matrix are linked through a linker arm containing a vicinal diol bond; vicinal diol hydroxyl is oxidized by a strong oxidant, so that a single bond adjacent to carbon is broken off and a fluorescent group is chemically cut simply and effectively; the same template to be sequenced is subjected to sequencing reactions in two groups, each sequencing is finished by two nucleotide dNTPs cycles marked by two pairs of different fluorescences; in a mode that each nucleotide can only be used once in one cycle, a code composed of a nucleotide fragment is obtained by each sequencing reaction, and nucleotide sequence information composed of a group of codes is obtained after the first group of sequencing reaction is finished; then the second group of sequencing reaction is performed to obtain a plurality of code information ranked by the second group of sequencing reaction; the two groups of code information are encoded according to the sequencing order, and the two groups of encoded information are combined to form the specific base sequence of a nucleotide fragment to be sequenced.

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 specially modified nucleotide and its application in high-throughput sequencing. Background technique [0002] DNA sequencing technology is the most commonly used technology in molecular biology research, and its appearance has greatly promoted the development of biology. Mature DNA sequencing technology began in the mid-1970s. In 1977, Maxam and Gilbert reported the method of determining DNA sequence by chemical degradation. During the same period, Sanger invented the dideoxy chain termination method. The fluorescent automatic sequencing technology that appeared in the early 1990s brought DNA sequencing into the era of automatic sequencing. These technologies are collectively referred to as first-generation DNA sequencing technologies. The second-generation DNA sequencing technology develope...

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Problems solved by technology

[0003] The three mainstream sequencing platforms have limitations. The biggest advantage of 454 sequencing technology lies in the longer read length. However, the cost of 454 sequencing technology is much higher than other sequencing technologies, which limits its application.

Due to the protection of the 3' end repressor group, only one base is extended each time, which increases the sequencing time, and due to the existence of the 3' end repressor group, its requirements for polymerase are very high, which also limits Increased its read length, increased its cost

[0004] In "DNA Sequencing Method for Simultaneous Synthesis of Two Nucleotides", a method and application for the simultaneous synthesis of two nucleotides to encode and decode nucleic acid sequences were proposed, but due to the high difficulty of nucleotide synthesis, the uncertainty is too large Large, there are no mature products on the market

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