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Whole genome amplification method based on slender reaction cavity

A whole-genome amplification and reaction chamber technology, which is applied in the field of efficient and high-uniformity amplification of whole-genome information, can solve problems such as poor amplification consistency, complicated experimental procedures, and complex systems, so as to avoid interference and simplify Response system, the effect of reducing operational complexity

Active Publication Date: 2017-08-18
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Technical problem: The purpose of the present invention is to address the current situation that the existing multiple strand displacement amplification method for the whole genome has poor consistency in the amplification of different regions of the genome, and the multiplex amplification method based on reaction microchambers and microdroplets. Strand Displacement Amplification method has complex system, high cost and complex experimental process. Through the transformation of the reaction chamber, the reaction vessel is converted into a slender continuous reaction chamber to improve the uniformity of amplification and simplify the system and experiment. process, reduce costs

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Genome-wide amplification of YH-1 immortalized cells using polytetrafluoroethylene (PTFE) capillary tubes.

[0023] Sample: 1ng human immortalized cell line YH-1 genomic DNA.

[0024] Reaction system: 2.5 nmol of N6 random sequence primer, 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, 10U; Φ29 DNA polymerase Enzyme reaction buffer, dilute to 50μl.

[0025] The reaction system was mixed on ice, and then injected into a polytetrafluoroethylene (PTFE) capillary with an inner diameter of 320 μm. The capillary was pre-filled with 0.1% (w / w) bovine serum albumin and treated at room temperature for 1 hour. After the reaction system is passed into the capillary, both ends of the capillary are sealed with epoxy resin glue, and the capillary is wound on a metal winding device. The cross-sectional area of ​​the capillary is about 80425 μm 2 , the square root of the cross-se...

Embodiment 2

[0028] Example 2: Amplification of the genome of a single TC-1 mouse immortalized cell using a microfluidic chip with a long and thin microchannel.

[0029] Sample: lysed mouse immortalized cell line TC-1 single cell.

[0030]Reaction system: 2.5 nmol of N9 random sequence primer, the sequence is 5'-NNNNNNNN-s-N-s-N-3', in which the 3' ends of the 7th and 8th bases are treated with sulfur modification; Klenow DNA polymerase, 5U; Klenow DNA polymerase reaction buffer, dilute to 20μl.

[0031] A glass microfluidic chip containing slender microfluidic channels is prepared by soft lithography. The microfluidic channels are repeatedly bent and arranged neatly. The cross-section of the microfluidic channels is rectangular, 400 μm wide, 500 μm deep, and 200 mm long. The bottom surface of the microfluidic chip is close to the semiconductor temperature control device for controlling the temperature of the chip.

[0032] The reaction system is mixed on ice, and then injected into the ...

Embodiment 3

[0035] Example 3: Using a stainless steel metal tube to amplify Arabidopsis genomic DNA.

[0036] Sample: 5ng Arabidopsis genomic DNA.

[0037] Reaction system: 2.5 nmol of N12 random sequence primer, the sequence is 5'-NNNNNNNNNNNN-3'; Bst DNA polymerase, 10 U; Bst DNA polymerase reaction buffer, dilute to 40 μl.

[0038] The reaction system was mixed on ice, and then poured into a stainless steel square tube with a square inner opening and a side length of 2 mm. The stainless steel tube was pre-filled with 0.1% (w / w) bovine serum albumin and treated at room temperature for 1 hour. The reaction system is passed into a stainless steel tube, and the two ends of the stainless steel tube are sealed with epoxy resin glue. The cross-sectional area of ​​the metal tube is 4mm 2 , the square root of the cross-sectional area is 2mm, and the length of the reaction zone is about 10mm. Subsequently, the stainless steel tube was placed in a water bath for 4 hours at 65°C, and then plac...

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Abstract

The invention provides a whole genome amplification method based on a slender reaction cavity; the method is characterized in that a multiple displacement amplification reaction system is injected into the slender reaction cavity so as to finish whole genome amplification for a target genome. The slender reaction cavity isolates the multiple displacement amplification reaction system relatively therein, material exchange and mutual influence of microscopic reaction units are greatly decreased, each microscopic reaction unit performs reacting in relatively independent state, and the uniformity of amplification between fragments of target nucleic acid is greatly improved; in addition, multiple displacement amplification based on the slender reaction cavity has no need for preliminarily preparing a micro-droplet generator or a complex reaction microcavity; trace liquid need not be precisely and complicatedly controlled during system debugging and amplification reaction, the reaction system is simplified, and operation complexity is decreased.

Description

technical field [0001] The invention relates to a technology for performing a whole genome amplification reaction by using a slender reaction chamber, which is a method for realizing efficient and high uniformity amplification of whole genome information, and belongs to the field of biotechnology. Background technique [0002] In the past 10 years, the method of whole genome amplification (WGA) using multiple strand displacement reaction (Multiple displacement amplification, MDA) (Genome research, 2001, 11: 1095-1099) has received great attention. The multiple strand displacement reaction can complete the effective amplification of low-initial samples, so that the quality and concentration of the amplified nucleic acid can meet the needs of various applications, especially the needs of constructing high-throughput DNA sequencing libraries. [0003] Different from another whole genome amplification technology that is also widely used—the polymerase chain reaction (Polymerase ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2531/119
Inventor 涂景陆祖宏李俊吉鲁娜乔祎
Owner SOUTHEAST UNIV
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