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

A technique for terminating nucleotides and single-molecule sequencing, applied in the field of genetic engineering, can solve the problems of slow extension, fluorescence quenching, high mismatch rate, etc., and achieve the effect of precise focusing

Active Publication Date: 2019-10-18
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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

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

Examples

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Embodiment 1

[0140] Example 1: Three-color fluorescent labeling reversible termination of nucleotide DNA single molecule sequencing system

[0141] The composition of the three-color fluorescent single-molecule sequencing system a1 in this embodiment is as follows: For the 3′-OH protected nucleotides without fluorescently labeled base G, it is composed of compound IV or VIII, and the corresponding fluorescently labeled base U nucleoside The acid is composed of one of compounds X, XIV, and XX; the corresponding fluorescently labeled base C nucleotide is composed of one of compounds XI, XVI, XXI, XXXVII, and the corresponding fluorescently labeled base A nucleotide is composed of XII, XVII, XXII, XXIII, XXXVIII; four modified nucleotides of different bases together form the sequencing reagent system 1 of this embodiment;

[0142] The composition of the reagent system a2 of the three-color fluorescent-labeled single-molecule sequencing system is as follows: For the 3′-OH protected nucleotides wit...

Embodiment 2

[0163] Example 2: Four-color fluorescent labeling reversible termination of nucleotide DNA single molecule sequencing system

[0164] In the four-color fluorescent single-molecule sequencing system of this embodiment, the reversible terminating nucleotide is selected in this embodiment. For base U, select XXVI, XXVII, XVIII, or XIX, for base C, select XXIX, for base G, XXXI or XXXII, select XXXV or XXXVI for base A, and four modified nucleotides of different bases together form the sequencing reagent system 1 of this embodiment;

[0165] Reagent system 2: compound X, XI, XII, XIII of the four-color fluorescent reversible termination nucleotide sequencing system of the present invention;

[0166] Reagent system 3: compound XIV, XVI, XVII, XV of the four-color fluorescent reversible termination nucleotide sequencing system of the present invention;

[0167] The four-color fluorescent reversible termination nucleotide sequencing system reagent system of the present invention 4: For U, s...

Embodiment 3

[0178] Example 3: Construction and application of single-molecule paired-end sequencing system

[0179] Use the sequencing chip of Example 1 to analyze the DNA template sequence to be tested (sequence 5)

[0180] 5'-GTTGTTGTTGTTGTTGTTCTACGTTCGAACTACTAAGCAATCCGGCAGATCGTCACAAAAAAAAAAAAAAAAAAAA-3' for double-ended single molecule sequencing. Specific steps are as follows:

[0181] (1) The primer immobilized on the surface of the substrate is 5'-(CNVC)TTUTTTTTTTTTTTTTTTTT-3' (sequence 6), where CNVC is a reversible light crosslinking agent between DNA strands, and then the template column to be sequenced is fixed with the The primers were incubated at 65°C for 5 minutes and slowly cooled to 37°C for the first hybridization; after the hybridization, four natural nucleotides (dATP, dTTP, dCTP, dGTP) were added, and the Under the action, the fixed primer is extended to synthesize a DNA strand complementary to the sequence of the template to be tested. Then the temperature was increased...

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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 ...

Claims

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Application Information

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IPC IPC(8): C12M1/38C12M1/34
CPCC12Q1/6869C12Q2533/101C12Q2563/107
Inventor 沈玉梅谭连江邵志峰龚兵李小卫
Owner SHANGHAI JIAO TONG UNIV
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