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Method for preparing molecular cloning chip for high-throughput cloning of nucleic acid molecule

A nucleic acid molecule and molecular cloning technology, applied in the field of high-throughput cloning of nucleic acid molecules and high-throughput molecular cloning chips, can solve the problems of high cost and small amount of templates for four-color fluorescence sequencing, and achieve easy hybridization and extension detection. Effect

Inactive Publication Date: 2011-02-09
SOUTHEAST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the throughput of the sequencing template chips prepared by Solexa is very high, the amount of templates they prepare is very small, and high-throughput sequencing can only be achieved through their four-color fluorescent sequencing method, but the cost of four-color fluorescent sequencing is extremely high, so It is the purpose of the present invention to improve the preparation method of sequencing templates, which can not only realize high-throughput sequencing, but also be applicable to low-cost sequencing methods

Method used

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  • Method for preparing molecular cloning chip for high-throughput cloning of nucleic acid molecule
  • Method for preparing molecular cloning chip for high-throughput cloning of nucleic acid molecule
  • Method for preparing molecular cloning chip for high-throughput cloning of nucleic acid molecule

Examples

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Effect test

Embodiment 1

[0025] A method for preparing a molecular cloning chip by high-throughput nucleic acid molecular cloning, the preparation steps are: a. Mixing the nucleic acid molecule template to be cloned with the nucleic acid amplification reaction system reagents to obtain an amplification mixture solution, so that each microliter of the amplification mixture solution The number of nucleic acid template molecules contained is 10 6 ~10 7 , the 5' end of the primer in the nucleic acid amplification reaction system reagent is modified with biotin, aldehyde group, acrylamide group or amino chemical group; b. adding a polymer compound to the above-mentioned amplification mixture solution, the polymer compound The compound can solidify the solution into a gel after lowering the temperature or heat treatment; c. adding twice its volume of the oil phase to the solution obtained in step b for emulsification to obtain a water-in-oil emulsion, the particle size of the emulsion particles 5 μm ± 2.5 ...

Embodiment 2

[0026] Example 2 Preparation of Nucleic Acid Molecular Cloning Array Using Emulsion PCR Amplification and Agarose Gel Particle Fixation

Embodiment approach

[0027] Concrete implementation scheme is as follows:

[0028] Preparation of genomic amplification templates with linkers on both sides:

[0029] Ultrasonic treatment of genomic DNA: choose an ultrasonic instrument with a power of 1200w, and randomly break the genomic DNA into gene fragments of 200bp-600bp size.

[0030] Both ends of genomic DNA fragments are connected with universal linkers: the random fragments obtained by ultrasound are purified to remove some small and broken fragments, and then T4 polynucleotide kinase, T4 DNA polymerase, and Klenow large fragment DNA polymerase are used respectively I and Taq polymerase treatment to make random fragments generate a TA sticky end, and then connect with a universal linker (linker1: 5'-GTCGGAGGCCAAGGCGGCCGT 3'linker2: 5'-CGCCTTGGCCTCCGACT-3') to connect both ends of the genomic fragment Two common linkers.

[0031] Amplification of the genome flanked by linkers in emulsion PCR with agarose and purification of agarose part...

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Abstract

The invention provides a method for preparing a molecular cloning chip by cloning high flux nucleic acid molecule. The method comprises the following steps: a. a cloning nucleic acid molecule template is mixed with a nucleic acid amplification reaction system reagent to obtain an amplification mixture solution; b. the amplification mixture solution is added with a high molecular compound; c. the solution is added with oil phase, of which the volume is two times of the volume of the solution for emulsification to obtain water-in-oil emulsion; d. the emulsion is placed in a PCR meter or a thermostat to undergo a nucleic acid amplification reaction; e. after the nucleic acid amplification reaction, the emulsion is cooled down to form nucleic acid molecule cloning grains; f. the nucleic acid molecule cloning grains are separated; and g. the nucleic acid molecule cloning grains are randomly dispersed on a solid phase carrier to fix the nucleic acid amplification products in the solution onthe solid phase carrier and form a nucleic acid molecule cloning array on the surface of the solid phase carrier and thus the cloning chip is obtained. The method has the advantages of improving preparation sequencing template, ensuring high flux of sequencing and lowering sequencing cost.

Description

1. Technical field [0001] The invention belongs to the technical field of molecular cloning in molecular biology, in particular to a method for high-throughput cloning of nucleic acid molecules, and the high-throughput cloned molecules are respectively fixed on solid-phase carriers to form a high-throughput molecular cloning chip. 2. Background technology [0002] Prior art: the human genome (sequencing) project has been completed, and the post-genome project has stepped into implementation. The study of the function of genes in the life process, that is, functional genomics, has become a common topic for life science workers all over the world. The purpose of functional genomics research is to understand the biological meaning of each nucleic acid in the genome and to discover disease susceptibility genes. The research approach of functional genomics is comparative genomics, that is, by comparing and analyzing the genome sequence differences between individuals with differ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/10
Inventor 周东蕊陆祖宏
Owner SOUTHEAST UNIV
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