Nanopore sequencing method for nucleic acid molecules with low perforation rate and special nanopore device thereof

Abstract

The invention discloses a nanopore sequencing method for nucleic acid molecules with low perforation rate and a special nanopore device thereof. The method comprises the following steps: adding to-be-detected nucleic acid molecules into a nanopore sequencing device filled with electrolyte, wherein the nanopore sequencing device comprises an electrolytic tank provided with a positive electrode and a negative electrode and a solid-state nanopore film which separates the positive electrode and the negative electrode of the electrolytic tank and covers an agarose gel layer; adding the nucleic acid molecules into a negative electrode chamber of the electrolytic tank; applying voltage between the positive electrode and the negative electrode during measurement to serve as a driving electric field of nucleic acid molecule perforation. The nanopore device consists of the solid-state nanopore film and the agarose gel layer covered by the film. According to the method disclosed by the invention, the signal to noise ratio of a measurement signal is not influenced, the motion speed of the nucleic acid molecules in the nanopores is effectively reduced, and the time resolution is improved; the high signal to noise ratio of the measurement signal is maintained, the problem that the signal to noise ratio is sacrificed to slow down DNA sequencing is solved, and a foundation is laid for realizing accurate DNA sequencing by utilizing solid-state nanopores; and the method is simple and easy to operate.

Description

technical field [0001] The invention relates to a nanopore sequencing method with low perforation speed of nucleic acid molecules and a special nanopore device, belonging to the field of material testing. Background technique [0002] DNA single-molecule detection and analysis based on solid-state nanopore devices is considered to be one of the most promising technical routes for realizing the third generation of rapid and low-cost human gene sequencing (realizing the genome sequencing of a single person within 24 hours at a cost of less than $1,000), and has become In addition to research groups such as Harvard University, Boston University, Brown University, Caltech, and Delft University of Technology in the Netherlands, IBM also announced to join the next generation of human genome sequencing in October 2009- The $1,000 plan uses nanopore device-based sequencing technology as a realization method, enabling nanopore research to move from basic research to application (D.Br...

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Problems solved by technology

Moreover, with the increase of glycerol, the viscosity coefficient of the solution increases, and the amplitude of the DNA perforation blocking current value decreases, so that the signal-to-noise ratio of the test decreases, resulting in an increase in the measurement error

In addition, the DNA perforation speed can be slowed down by lowering the voltage, but the lowering of the voltage leads to a lower current signal and a worse signal-to-noise ratio, making measurement very difficult

It can be seen that many existing methods of slowing down the speed of DNA perforation are only possible on the basis of sacrificing the signal-to-noise ratio, which makes it very difficult to measure the signal accurately, and it is not easy to obtain accurate relevant biological information

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