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A Whole Genome Amplification Method Based on Agarose Gel Medium

A whole-genome amplification and agarose gel technology, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of high cost, high dependence, high price, etc. Insufficient amplification, improved amplification uniformity, and reduced operational complexity

Active Publication Date: 2022-01-28
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the preparation and operation of the reaction microcavity is relatively complicated, relying on sophisticated equipment and delicate operation, and the cost is relatively high. It is time-consuming and labor-intensive to construct the entire system from scattered materials and equipment.
The transfer of multiple strand displacement amplification into an oil-water mixed emulsion environment is also extremely dependent on equipment and operations. The price of commercial microdroplet generator machine accessories is relatively high, and the surfactants used to prepare microemulsions are limited to a certain extent. affect the reaction

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Expansion of YH-1 Immortalized Cells Using Agarose Gel Medium

[0025] Sample: 10ng human immortalized cell line YH-1 genomic DNA.

[0026] Add 5 nmol of N6 random sequence primers to the PCR centrifuge tube, the sequence is 5'-NNNN-s-N-s-N-3', in which the 3' ends of the 4th and 5th bases are treated with sulfur modification, Φ29 DNA polymerase reaction buffer , and set the volume to 90 μl. Prepare a 5% (w / v) low-melting point agarose aqueous solution, melt it into a transparent liquid at high temperature, take 10 μl and add it to the reaction system, and the final mass-volume ratio of the agarose in the reaction system is 0.5%. After the reaction system was cooled to 60°C, 10 UΦ29 DNA polymerase was added, and continued to cool at room temperature for 40 minutes until the reaction system turned into a gel state. Place the centrifuge tube in a 30°C water bath and incubate for 16 hours, then put it in another 65°C water bath for 5 minutes to inactivate Φ29 ...

Embodiment 2

[0029] Example 2: Using agarose gel medium to amplify the genome of a single TC-1 mouse immortalized cell

[0030] Sample: lysed mouse immortalized cell line TC-1 single cell.

[0031]Add 5 nmol of N9 random sequence primers with the sequence 5'-NNNNNNNNNN-3'Klenow DNA polymerase reaction buffer to the PCR centrifuge tube, and make the volume to 40 μl. A 5% (w / v) agarose aqueous solution was prepared, and after being melted into a transparent liquid at high temperature, 10 μl was added to the reaction system. The final mass-volume ratio of the agarose in the reaction system was 1%. After the reaction system was cooled to 60°C, 5U Klenow DNA polymerase was added, and continued to cool at room temperature for 40 minutes until the reaction system turned into a gel state. Place the centrifuge tube in a 37°C water bath for 16 hours, and then place it in another 65°C water bath for 5 minutes to inactivate Klenow DNA polymerase.

[0032] After the reaction was completed, the reacti...

Embodiment 3

[0034] Example 3: Amplification of Arabidopsis genomic DNA using agarose gel medium.

[0035] Sample: 15ng Arabidopsis genomic DNA.

[0036] Add 7.5nmol of N12 random sequence primers to the PCR centrifuge tube, the sequence is 5'-NNNNNNNN-s-NN-s-NN-3', wherein the 3' ends of the 8th and 10th bases are treated with sulfur modification; Bst DNA polymerase reaction buffer, dilute to 10μl. A 5% (w / v) agarose aqueous solution was prepared, and after being melted into a transparent liquid at high temperature, 10 μl was added to the reaction system. The final mass-volume ratio of the agarose in the reaction system was 2.5%. After the reaction system was cooled to 70°C, 10U of Bst DNA polymerase was added, and continued to cool at room temperature for 45 minutes until the reaction system turned into a gel state. Put the centrifuge tube into the PCR amplification instrument, and the program is set to 65°C for 4 hours and 80°C for 20 minutes.

[0037] After the reaction was complete...

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PUM

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Abstract

The invention discloses a whole-genome amplification method based on an agarose gel medium, which comprises placing a multi-strand displacement amplification reaction system in the agarose gel, and completing the target genome amplification in the grid-like structure of the gel. whole genome amplification. The agarose in the gel state forms a grid structure, which relatively separates the multi-strand displacement amplification reaction system in a small reaction space, and greatly reduces the material exchange and mutual influence between each microscopic reaction unit, so each microscopic reaction unit The reaction occurs in a relatively independent state, and the amplification consistency between each fragment of the target nucleic acid is greatly improved. At the same time, multiple strand displacement amplification based on agarose gel medium does not require pre-preparation of micro-droplet generating devices or complex reaction micro-cavities, and in the process of system debugging and amplification reactions, there is no need for precise and accurate monitoring of micro-fluids. Complex control simplifies the reaction system and reduces the complexity of operation.

Description

technical field [0001] The invention relates to a whole genome amplification method based on an agarose gel medium, belonging to the technical field of whole genome amplification. Background technique [0002] Whole genome amplification (WGA) is an amplification method to increase the number of limited DNA samples. Early WGA was based on polymerase chain reaction (Polymerase Chain Reaction, PCR) technology, such as degenerate oligonucleotide PCR (DOP–PCR), primer extension preamplification (PEP) and adapter ligation PCR, etc. . Taq DNA polymerase, used in these techniques, limits fragment size to 3kb and introduces many errors into the sequence. Furthermore, these techniques do not provide complete coverage of the genome, and amplification is biased. [0003] In the past ten years, multiple displacement amplification (MDA), which combines random hexamers with denatured DNA and then uses Phi29 polymerase to perform strand displacement synthesis at a constant temperature, h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2531/119
Inventor 涂景苏奥马靖原黄梦婷杨芳陆祖宏
Owner SOUTHEAST UNIV
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