DNA synthesis sequencing method and system

Abstract

The invention discloses a DNA synthesis sequencing method and a DNA synthesis sequencing system. The method comprises the following steps: connecting a primer P1 to the surface of a matrix through a water-soluble bifunctional linking unit; adding a solution containing a DNA template to be detected onto the surface of the matrix, and carrying out isothermal surface amplification reaction; adding aprimer Pe to the surface of the matrix, adding an extension reaction solution containing fluorescence labeling reversible terminators of four basic groups onto the surface of the matrix, and carryingout extension reaction; carrying out imaging on DNA double strands after extension, and adding a corresponding chemical reagent, so that the linking units of the fluorescence labeling reversible terminators break; and repeating the steps of DNA chain extension and breakage, and completing a plurality of times of cycle sequencing, thus obtaining the base sequence of nucleotide of the DNA template to be detected. The sequencing method provided by the invention can be used for DNA high-throughput synthesis sequencing, according to the experimental result, under the sequencing condition, the readlength of sequence is at least 73 base groups, if double-end sequencing is adopted, the read length is at least 146, and the accuracy rate is at least 99.98%.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a DNA synthesis sequencing method and a sequencing system. Background technique [0002] With the completion of the Human Genome Sequencing Project, DNA sequencing technology has developed rapidly. DNA sequencing (DNAsequencing) refers to the analysis of the base sequence of a specific DNA fragment, that is, the sequence of adenine (A), thymine (T), cytosine (C) and guanine (G). The development of accurate, high-throughput, and low-cost DNA sequencing methods is of great significance for biology and medicine. [0003] Sequencing By Synthesis (SBS) is one of the second-generation DNA sequencing technologies. Sequencing by synthesis fixes a large number of template DNA fragments to be tested, and hybridizes on the fixed DNA sequencing template to combine with universal DNA primers, and then four base fluorescently labeled nucleotides are sequentially extended and brok...

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Problems solved by technology

[0005] First of all, the synthesis of modified nucleotides protected by 3'-OH is complicated; secondly, in the actual sequencing process, after extension using a reversible terminator protected by 3'-OH, the two reaction sites need to be broken at the same time, that is, the linking unit is 100% broken It is also necessary to deprotect the 3'-OH with 100% efficiency

However, these two sites often cannot be completely broken at the same time under the same conditions, which will inevitably lead to the accumulation of error information, which will eventually affect the read length and efficiency of sequencing

However, DNA sequencing needs to be 100% removed. When the modified nucleotides protected by the 3'-OH participate in DNA chain extension, another huge challenge is that DNA polymerase is not easy to recognize, the extension reaction is slow, and it is easy to mismatch (Ref:Michael L . Metzker, Nucleic Acids Research 2012, 40, 7404; Michael L. Metzker; Nature Reviews Genetics 2010, 11, 31.)

[0006] Reversible terminators based on disulfide linkage units have been applied in both next-generation sequencing and single-molecule sequencing. In order to ensure that the disulfide bond reversible terminator is used as a single-molecule sequencing reagent to extend only one reversible terminator at a time, Helicos has connected a nucleus with a large steric hindrance next to fluorescein. Glycoside monophosphate or diphosphonic acid, the reversible terminator with such a large steric hindrance can indeed extend one at a time, but its synthetic route is extremely complicated, and the excessive steric hindrance also makes it difficult for enzymes to recognize when participating in DNA chain elongation And slow speed, high mismatch rate (Michael L.Metzker; Nature Reviews Genetics 2010,11,31.)

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