DNA single-molecule sequencing system and device based on multicolor fluorescent reversible terminating nucleotides
A technology for terminating nucleotides and single-molecule sequencing, applied in the field of genetic engineering, can solve problems such as high mismatch rate, complicated synthetic route, slow extension speed, etc., to reduce the probability of mismatch, GC preference, and error rate Effect
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Embodiment 1
[0140] Example 1: Three-color fluorescence-labeled reversible terminating nucleotide DNA single-molecule sequencing system
[0141] The composition of the three-color fluorescent single-molecule sequencing system a1 described in this example is as follows: for the 3′-OH protected nucleotide 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 the compounds X, XIV, XX; the corresponding fluorescently labeled base C nucleotide is composed of one of the compounds XI, XVI, XXI, XXXVII, and the corresponding fluorescently labeled base A is composed of XII, XVII, XXII, XXIII, XXXVIII; four modified nucleotides with different bases together constitute the sequencing reagent system 1 of this embodiment;
[0142] The reagent system a2 of the three-color fluorescently labeled single-molecule sequencing system is composed as follows: for the 3′-OH protected nucleotide wi...
Embodiment 2
[0163] Example 2: Four-color fluorescence-labeled reversible terminating nucleotide DNA single-molecule sequencing system
[0164] In the four-color fluorescent single-molecule sequencing system described in this embodiment, in this embodiment, the reversible termination nucleotide is selected as XXVI, XXVII, XVIII, or XIX for base U, XXIX for base C, and XXIX for base G. For XXXI or XXXII, select XXXV or XXXVI for base A, and the modified nucleotides of four different bases together form the sequencing reagent system 1 of this embodiment;
[0165] Reagent system 2 of the four-color fluorescence reversible termination nucleotide sequencing system of the present invention: compounds X, XI, XII, XIII;
[0166] Reagent system 3 of the four-color fluorescence reversible termination nucleotide sequencing system of the present invention: compounds XIV, XVI, XVII, XV;
[0167] Reagent system 4 of the four-color fluorescent reversible termination nucleotide sequencing system of the...
Embodiment 3
[0178] Example 3: Construction and application of single-molecule paired-end sequencing system
[0179] Adopt the sequencing chip of embodiment 1 to DNA test template sequence (sequence 5)
[0180] 5'-GTTGTTGTTGTTGTTGTTCTACGTTCGAACTACTAAGCAATCCGGCAGATCGTCACAAAAAAAAAAAAAAAAAAAA-3' for paired-end 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), wherein CNVC is a reversible photocrosslinking agent between DNA strands, and then the template sequence to be sequenced and the immobilized 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, at 37°C, DNA polymerase Under the action, the fixed primer is extended to synthesize a DNA strand complementary to the sequence of the template to be tested. Then heat up to 65°C fo...
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Abstract
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