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Single molecular nucleic acid sequencing process for exonuclease-nanometer hole

A molecular nucleic acid and nanopore technology, which is applied in the field of exonuclease-nanopore single-molecule nucleic acid sequencing, can solve the problems of unusable sequencing, intolerance, and high error rate, and achieve high signal-to-noise ratio, improved accuracy, and high The effect of voltage

Inactive Publication Date: 2007-03-21
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because protein nanopores are used, they cannot withstand high voltage, are prone to aging, and the error rate is too high, so they cannot be used for sequencing

Method used

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  • Single molecular nucleic acid sequencing process for exonuclease-nanometer hole

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: DNA Fragment Sequencing

[0018] ① Detection of dNMPs handwriting

[0019] Add electrophoresis fluid to the two-stage electrophoresis tank connected to the patch clamp, separate the electrophoresis tank from the middle with nano-components with controllable diameter and hole depth, add only one kind of ultra-diluted nucleotide each time, turn on the power and record The electrical signal when a single nucleotide passes through the nanopore; in this way, the perforation electrical signal of various nucleotides is detected, and finally their specific handwriting is determined, a standard curve is established, and software is written.

[0020] ②Calculate the swimming speed of nucleotides, and find the farthest exonuclease action point of the base sequence;

[0021] When each of the above detections is over, the positive and negative electrodes are switched to each other, so that the nucleotides gathered on the original positive electrode migrate to the new posi...

Embodiment 2

[0032] Example 2: Complete Chromosome Sequencing

[0033] ①Single-molecule operation: In this example, the nucleotide handwriting detection and the farthest point of action of the exonuclease are the same as in Example 1.

[0034] A single chromosome is extracted with the aid of a microscope, flow cytometer or other methods, and the chromosome is immobilized on magnetic beads according to the method in Example 1.

[0035]② Chromosome fixation: fix the magnetic beads with 1 complete chromosome on the magnet installed on the negative end of the electrophoresis tank of a sequencing unit. Each sequencing unit has a chromosome.

[0036] ③Sequence detection: Add exonuclease to the negative electrode of the electrophoresis tank. After a single enzyme molecule binds to DNA, add magnesium ions, turn on the power immediately, collect electrical signals, and use software to read the DNA sequence.

[0037] ④ Detection of complementary chain sequence: same as in Example 1.

[0038] Effe...

Embodiment 3

[0039] Example 3: RNA sequencing

[0040] ① The detection of rNMPs handwriting is the same as in Example 1, and a standard curve is established according to the specific electrical signals generated by them.

[0041] ②According to the method of Example 1, RNA single molecules were fixed to the negative electrode of the electrophoresis tank, nucleotides were degraded one by one with RNA exonuclease, the handwriting was recorded by patch clamp, and the electrical signal was converted into sequence information according to the standard curve.

[0042] ③ RNA can also be used as a template to synthesize cDNA (complementary DNA) by reverse transcriptase, and then obtain the sequence of RNA according to the steps in Example 1.

[0043] Implementation effect: suitable for the RNA / DNA exonuclease activity of the present invention at 1000nts / second, such as detection of mRNA sequence, according to the average length of mRNA of 1000nts, the array of 100×100, that is, 10000 mRNA or cDNA p...

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Abstract

The present invention relates to biotechnology, and is single molecular nucleic acid sequencing process for exonuclease-nanometer hole. The process includes the following steps: 1. detecting electric signal of single molecular nucleotide to set up standard curve; 2. fixing nucleic acid in the negative pole of electrophoresis tank, degrading nucleotides with exonuclease and detecting the electric signal with the patch clamp while passing through the nanometer hole; and 3. converting the electric signal into specific nucleotide based on the standard curve and obtaining the base sequence of the nucleotide according to the cutting direction of the exonuclease. The present invention has high nucleoside and deoxynucleoside-phosphate recognizing accuracy, and may be used in detecting nucleoside and deoxynucleoside5'-phosphate, nucleoside and deoxynucleoside3'-phosphate and methylated cytidine in natural eukaryote DNA.

Description

technical field [0001] The invention relates to a determination method in the field of biotechnology, in particular to an exonuclease-nanopore single-molecule nucleic acid sequencing method. Background technique [0002] With the advent of the post-genome generation, the demand for the sequencing of biological macromolecules such as DNA and RNA has increased significantly, but the current sequencing methods are slow, expensive, and have gaps, which seriously hinder the development of related disciplines, such as genome sequencing, According to the results released by NCBI in June 2006, 21 eukaryotic genomes have been sequenced, 100 have been assembled, and 164 are in progress. In fact, this is just the prelude to a large number of genome sequencing, because the need for genome sequencing is manifold. However, it is still very unrealistic to use the current method to meet the above-mentioned multiple sequencing requirements. The main reason is that the current method has man...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王志民
Owner SHANGHAI JIAO TONG UNIV
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