Nanopore based dislocation sequencing method for direct sequencing of non-natural nucleic acid

Abstract

The invention discloses a nanopore based dislocation sequencing method for direct sequencing of non-natural nucleic acid. The method includes the steps of: (1) conducting solid-phase synthesis of a non-natural nucleic acid to-be-sequenced chain, connecting the non-natural nucleic acid to-be-sequenced chain with a DNA-guided sequencing chain by enzymatic ligation to serve as a complete to-be-sequenced chain; (2) preparing a nano-sensing pore channel with single-molecule resolution ability; and (3) mixing the to-be-sequenced chain with a DNA primer, conducting denaturing annealing to construct asequencing library, and then adding the library and nucleic acid polymerase into a cis chamber of the nanopore together, under the action of an electric field force, passing the nucleic acid chain through the nanopore, reading the electrical signal generated when the nucleic acid chain passes through the pore, and analyzing the signal to obtain the sequence. Compared with the prior art, the invention utilizes a low-cost and high efficiency nanopore sequencing method to realize direct sequencing of the continuous unnatural nucleic acid chain for the first time.

Description

technical field [0001] The invention relates to a dislocation sequencing method for direct sequencing of unnatural nucleic acids based on nanopores, belonging to the technical field of unnatural nucleic acid sequencing. Background technique [0002] Non-natural nucleic acids refer to nucleic acid analogs with chemical modifications. FANA (2'-deoxy-2'-fluoroarabinonucleic acid, 2'-fluoroarabinose nucleic acid) belongs to a class of unnatural nucleic acids. Compared with natural nucleic acids, FANA is more resistant to acid degradation and enzyme degradation. Because of its strong affinity with RNA, FANA is widely used in the field of gene therapy. Masad J. Damha team reported in 2006 that the antisense PS-FANA-DNA chimera has better resistance to degradation in living cells. In the same year, they compared the gene silencing efficiency of FANA-modified siRNA and natural siRNA molecules, and found that siRNA containing FANA had better performance in triggering RNA interferen...

AI Technical Summary

Problems solved by technology

However, there is a defect in this indirect sequencing method: because there is a certain error rate in the nucleic acid polymerization reaction catalyzed by enzymes, the error rate of this indirect sequencing method is not only derived from the errors in the DNA sequencing process, but actually includes FANA reverse transcription is the superposition of error rates in the two steps of DNA and DNA amplification. These factors will interfere with the determination of the final sequence. Therefore, this study is dedicated to finding a technology that can directly sequence FANA

[0005] However, at present, nanopore sequencing technology is mostly applied to natural nucleic acids, and there is no method for sequencing continuous non-natural nucleic acid chains.

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