Detection method for biomolecular probe-calibrated specific sites of DNA based on nanopore device

Abstract

The invention discloses a detection method for biomolecular probe-calibrated specific sites of DNA based on a nanopore device and application thereof. The method comprises the following steps: 1) calibrating the specific sites of DNA by using a biomolecular probe so as to obtain calibrated DNA; and 2) detecting the specific sites of the calibrated DNA by using a nanopore sequencing device, wherein the calibrated DNA is added into a positive electrode chamber filled with an electrolyte, an external pressure field is introduced to the positive electrode chamber during detection and is used as an external driving field for DNA perforation, voltage is applied between a positive electrode and a negative electrode and used as an external electric field with a direction opposite to the direction of the external pressure field, and an active force of the external pressure field on the DNA in a nanopore during detection is greater than an electric field force. With the method provided by the invention, secondary current signals totally different from those of common unmodified DNA are obtained, the spatial positions and sequence of modified specific sites of the DNA can be characterized through analysis and statistics of the secondary current signals, so a physical map of the long chain DNA is obtained.

Description

technical field [0001] The invention relates to a method for detecting specific sites of DNA marked by biomolecular probes based on a nanopore device. 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), becoming 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.Branton, et al.Nature Biotechnology 26(10), 1146(2008); M.Zwol...

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Problems solved by technology

It should be pointed out that each small fragment of hundreds of bp is relative to the whole human genome (about 3×10 9 bp) is too small, so the amount of calculation and algorithm complexity in the comparison and splicing of small fragments is extremely high, and because there are a large number of repetitive sequences in the human genome, the whole genome obtained by existing algorithms There is still an uncertainty of 5% to 10%. Therefore, for medical diagnosis, drug screening, and high-end scientific research fields that require high precision, it is necessary to repeat the entire sequence for more than a dozen or even hundreds of times, which undoubtedly greatly increases the time required for sequencing. cost and reduced time efficiency

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