DNA single-molecule sequencing system and apparatus based on multicolor-fluorescence reversible termination nucleotide

Abstract

The invention provides DNA single-molecule sequencing system and apparatus based on multicolor-fluorescence reversible termination nucleotide. The DNA single-molecule sequencing system comprises a primer, a DNA template to be tested and multicolor-fluorescence reversible termination nucleotide reagents. The primer is fixed on a surface of a flow cell reactor; after hybridization of the sequencingprimer with the DNA template to be tested, the primer is extended by using the multicolor-fluorescence reversible termination nucleotide reagents; and thus, sequence information of the DNA template tobe tested can be obtained by detecting fluorescence signals of the extended primer. Localizing fluorescent marker is not required for the 3'-terminal of the DNA template to be tested. By extending fluorescence of a reactant as localizing fluorescence of the next extension in sequencing cycle or adopting a localizing fluorescent marker fixed on the surface of the flow cell reactor as localizing fluorescence, the DNA single-molecule sequencing system requires no localizing fluorescent marker at the 3'-terminal of the DNA template to be tested; so that, the problem of location information loss caused by quenching is effectively avoided. Thus, sequencing reading length can be further and greatly extended with error rate reduced.

Description

Technical field [0001] The present invention relates to the field of genetic engineering, in particular to a DNA single molecule sequencing method and device based on multicolor fluorescent reversible terminating nucleotides. Background technique [0002] After the completion of the Human Genome Project, DNA sequencing technology has developed rapidly. DNA sequencing (DNAsequencing) refers to the analysis of the base sequence of specific DNA fragments, 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 to biology and medicine. [0003] DNA sequencing by synthesis second-generation sequencing technology has been widely used, but its inherent limitations are also obvious. For example, sequencing time is long, DNA amplification may introduce a certain error rate, etc. Therefore, the single-molecule-based third-generation sequencing technology has ...

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Problems solved by technology

Reversible terminators based on disulfide linkage units have been applied in single-molecule sequencing. However, the literature (Nucleic Acids Research, 2008, 36, No. 4e25) reported that reversible terminators based on disulfide bonds were single-color fluorescently labeled four Nucleotides with different bases, 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, a highly hindered nucleoside monophosphate or diphosphate is connected next to fluorescein. Phosphonic acid inhibitors, this type of reversible terminator can indeed extend only one sequencing cycle, but its synthesis route is complicated, and at the same time, the large steric hindrance leads to slow extension and high mismatch rate when participating in DNA chain extension (Michael L. Metzker; Nature Reviews Genetics 2010, 11, 31.)

[0007] Moreover, in the prior art, in order to perform localization, it is often necessary to mark specific localization fluorescence information on the 3' end of the template to be tested, and then to perform sequencing fluorescence detection on the primer / template complex after participating in the extension reaction. The fluorescein labeled at the 3' end of the template to be tested is irradiated and excited to locate the primer / template complex. The localized fluorescence information needs to be irradiated and excited during each extension reaction, and multiple repeated excitations are likely to cause its Fluorescence quenching, resulting in the loss of localization information, which ultimately leads to short read lengths in single-molecule sequencing

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