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Nucleotide sequence analysis method based on magnetic separation and high-fidelity polymerase correction function

A high-fidelity polymerase and nucleic acid sequence analysis technology, which is applied in biochemical equipment and methods, and the determination/inspection of microorganisms. It can solve the problems of insufficient read length and low background signal, so as to reduce background noise and signal accumulation. effect, the effect of great application prospects

Active Publication Date: 2011-09-21
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Purpose of the invention: The purpose of the present invention is to solve the problem that the read length is not long enough in the traditional Sanger sequencing method mentioned above, and propose a nucleic acid sequence analysis method based on magnetic separation and high-fidelity polymerase correction function, which has low background signal, Advantages of longer read length and ease of operation

Method used

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  • Nucleotide sequence analysis method based on magnetic separation and high-fidelity polymerase correction function
  • Nucleotide sequence analysis method based on magnetic separation and high-fidelity polymerase correction function
  • Nucleotide sequence analysis method based on magnetic separation and high-fidelity polymerase correction function

Examples

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Embodiment 1

[0036] Example 1 Preparation of gold magnetic nanoparticles suitable for sequencing

[0037] Magnetic nanoparticles refer to materials with at least one dimension between 1-1000 nm in particle size. When they reach a certain critical size, they will have a single magnetic domain and exhibit superparamagnetism. It has been gradually produced and developed since the 1970s. New biomedical functional materials developed. Under normal circumstances, the magnetic particles in the colloidal state are in a disorderly state of motion, can be stably suspended in the liquid medium, and conveniently carry out various biological reactions; and under the action of an external magnetic field, the magnetic particles show good magnetic properties. Responsiveness, the particles can be easily separated from the medium; when the external magnetic field is removed, the magnetic particles can be resuspended in the liquid medium without aggregation. In the embodiment of the present invention, the f...

Embodiment 2

[0040]Example 2 Verification of ddNTP correction function by Pfu high-fidelity DNA polymerase

[0041] The 3'-5' exonuclease activity is an intrinsic function of many DNA polymerases. It checks the incorporated nucleotides during DNA synthesis and removes nucleotides that do not match the template from the 3'-5' direction. Nucleotides are called the proofreading function of DNA polymerase. According to the different needs of DNA synthesis, various DNA polymerases have been transformed by modern genetic engineering methods, and DNA polymerases with different functions have been obtained. Pfu high-fidelity DNA polymerase is one of them. Pfu high-fidelity DNA polymerase can not only remove nucleotides that do not match the template incorporated during DNA synthesis from the 3'-5' direction, but also remove hindered nucleotides incorporated during DNA synthesis A chain-continuing nucleotide, such as a modified nucleotide with steric hindrance or a ddNTP that terminates the elonga...

Embodiment 3

[0043] Example 3 Sequencing of p-GEM-T Easy plasmid vector based on the principle of nucleic acid sequence analysis based on magnetic separation and high-fidelity polymerase correction function

[0044] By modifying streptavidin on the gold magnetic nanoparticles obtained in Example 1, the following sequencing process can be performed:

[0045] ① Design four primers, the first base downstream of which is one of the known A, T, C, and G, and the 5' end is modified with biotin, and the primers are fixed on the Streptavidin-gold magnetic nanoparticles. Cy5-labeled dideoxyribonucleotide monomers ddATP, ddTTP, ddCTP, and ddGTP were divided into four groups for single-base extension reactions in parallel, and the signal values ​​were recorded after scanning with a scanner equipped with Cy5 color filters as follow-up signals Processing benchmarks.

[0046] ② According to the SP6 site of the p-GEM-T Easy plasmid vector, the sequencing primer was designed as follows: TTCTATAGTGTCACCT...

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Abstract

The invention discloses a nucleotide sequence analysis method based on a magnetic separation and high-fidelity polymerase correction function. The method comprises the following steps of: 1) fixing a sequencing primer digested by a nucleic acid resistant exonuclease on a magnetic medium and hybridizing the sequencing primer with a DNA sequence to be detected; 2) adding an extension reaction solution of which the component comprises a DNA polymerase which does not have the activity of the exonuclease, extending a marked deoxyribonucleotide monomer and a marked bideoxyribonucleotide monomer, and obtaining the type information and the number information of nucleotides by detecting a marked object signal; 3) adding an extension reaction solution of which the component comprises the DNA polymerase which has the activity of the exonuclease, removing the extended dideoxy nucleotide (ddNTP) in step 2) through the correction function of a high-fidelity polymerase, and substituting and extending the nucleotide monomer digested by the nucleic acid resistant exonuclease; and 4) performing magnetic separation and washing on the magnetic medium, and circulating the step 2), the step 3) and the step 4) until the nucleotide sequence information in the DNA to be detected is determined.

Description

[0001] technical field [0002] The present invention relates to the field of nucleic acid sequence analysis, in particular to a nucleic acid sequence analysis method based on magnetic separation and high-fidelity polymerase correction functions, and the nucleic acid sequence is longer than the sequence length that can be analyzed by conventional Sanger sequencing methods. Background technique [0003] The genetic information of organisms is stored in the form of chromosomes organized by very long deoxyribonucleic acid (DNA) molecules. In biological research, DNA sequence analysis is crucial for the acquisition of genetic information and is the basis for the development of genetics and genomics. In-depth and comprehensive genetics and genomics research based on sequencing technology can be applied to basic research (identification of gene function, construction of signaling pathways, systems biology, biological evolution, research on disease mechanism, etc.), and can also ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 何农跃曾新
Owner SOUTHEAST UNIV
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