Method for linking monomolecular DNA to single magnetic bead

A linked, single-molecule technology, applied in the field of DNA processing, can solve the problems of cumbersome steps and low efficiency, and achieve the effect of improving sequencing throughput

Inactive Publication Date: 2011-07-27
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires equipment such as optical tweezers, and the steps are cumbersome and the efficiency is relatively low.

Method used

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  • Method for linking monomolecular DNA to single magnetic bead
  • Method for linking monomolecular DNA to single magnetic bead

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Step 1, the DNA is fluorescently labeled with YOYO-1, then the 3' end of the DNA is labeled with biotin, and the 5' end is labeled with digoxin;

[0021] Step 2, placing the DNA on the glass whose surface is coated with anti-digoxigenin, so that the 5' end of the DNA reacts with the anti-digoxigenin on the glass surface to immobilize on the surface, and the unfixed DNA molecules are washed away with running water;

[0022] Step 3: Add anti-streptavidin-coated micron or nano-magnetic beads to the glass surface, so that the anti-streptavidin on the magnetic beads reacts with the biotin at the 3' end of the DNA, and washes away the unconnected DNA. free magnetic beads; at this time, the 3' end of the DNA molecule on the glass surface is connected to the glass surface, and the 5' end is connected to the magnetic bead;

[0023] Step 4: Use a micropistol tip to absorb an appropriate amount of DNA endonuclease mixture, and with the assistance of a fluorescent microscope, add t...

Embodiment 2

[0030] Step 1: Fluorescently label the DNA with ethidium bromide, then label its 3' end with biotin, and its 5' end with digoxigenin;

[0031] Step 2: Place the DNA on the glass surface coated with anti-streptavidin, make the 3' end biotin of the DNA react with the anti-streptavidin on the glass surface to immobilize on the surface, wash off the unfixed DNA with running water DNA molecules;

[0032] Step 3: Add anti-digoxigenin-coated micron or nano-magnetic beads to the glass surface, so that the anti-digoxigenin on the magnetic beads reacts with the digoxin at the 5' end of the DNA, and washes away the DNA that is not connected to the DNA. free beads. At this time, the 5' end of the DNA molecule on the glass surface is connected to the glass surface, and the 3' end is connected to the magnetic beads. Only a very small number of magnetic beads are connected to more than one DNA molecule, which can be identified by rotating the magnetic bead with a magnetic bar, because a si...

Embodiment 3

[0038] Step 1, the DNA is fluorescently labeled with Hoechst 33342, then the 3' end is labeled with digoxigenin, and the 5' end is labeled with biotin;

[0039] Step 2: Place the DNA on the glass surface coated with anti-digoxigenin, make the 3' end of the DNA react with the anti-digoxigenin on the glass surface to immobilize on the surface, and wash away the unfixed DNA molecules;

[0040] Step 3: Add anti-streptavidin-coated micron or nano-magnetic beads to the glass surface, so that the anti-streptavidin on the magnetic beads reacts with the biotin at the 5' end of the DNA, and wash with running water to remove the unconnected DNA. free magnetic beads. At this time, the 3' end of the DNA molecule on the surface is connected to the glass surface, and the 5' end is connected to the magnetic beads;

[0041] Step 4: Use a micropistol tip to absorb an appropriate amount of DNA endonuclease mixture, and with the assistance of a fluorescent microscope, add the enzyme digestion m...

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PUM

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Abstract

The invention relates to a method for linking monomolecular DNA to a single magnetic bead, belonging to the technical field of biology and comprising the following steps: performing fluorescence labeling to DNA, then labeling biotin at the end 3' of the DNA and labeling digoxin at the end 5' of the DNA; or labeling digoxin at the end 3' of the DNA and labeling biotin at the end 5' of the DNA; placing the DNA on glass to carry out an immunoreaction and washing the DNA by running water; carrying out the immunoreaction again; adding DNA endonuclease to the periphery of magnetic beads, after DNA molecules are degraded, collecting magnetic beads carrying DNA fragments one by one by a micro-gunpoint and placing the magnetic beads on a magnet; and cutting target DNA by the endonuclease and linking target DNA fragments with the DNA fragments on the magnetic beads under the action of ligase. The method for linking the monomolecular DNA to the single magnetic bead can acquire the sequence information of target fragments and realize the monomolecular sequencing of the DNA; and when nano-pores are produced into an array to be matched with a decoy array, sequencing throughput can be improved.

Description

technical field [0001] The invention relates to a DNA processing method in the field of biotechnology, in particular to a method for connecting single-molecule DNA to a single magnetic bead. Background technique [0002] The basic operation of single-molecule sequencing is: use a film with a single nanopore sensor to divide the electrophoresis tank into two poles, in the negative electrode electrophoresis solution, fix one end of a nucleic acid fragment to be tested on a support, and use exonucleic acid cutting The enzyme degrades the nucleic acid components (4 kinds of bases) one by one from the free end. They are negatively charged at neutral to slightly alkaline pH. Under the action of an external electric field, the released bases are in the order of the original nucleic acid. When moving from the negative electrode to the positive electrode, it passes through the nanopore. Because the molecular weight and three-dimensional structure of the four bases are different, when...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 王志民李明
Owner SHANGHAI JIAOTONG UNIV
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