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Micro-volume cell nucleic acid amplification method

A cell nucleic acid, micro-volume technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as being unsuitable for popularization, low throughput, and inability to be popularized

Active Publication Date: 2018-07-24
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reaction volume is on the order of tens to hundreds of nanoliters, but the device is relatively complicated, needs to be driven by different pressure sources, and requires trained experimenters to operate, and the processing of the chip is also relatively complicated, with high cost and low throughput , not suitable for popularization
[0005] In short, the existing technology is complex in operation, high in cost and low in flux, and cannot be popularized

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0046] figure 1 Among them, the inner diameter of the quartz capillary 1 is 50 microns, the outer diameter is 150 microns, and the length is 5 cm. The upper end of the quartz capillary 1 is connected with a syringe pump (Harvard Apparatus, Pico Elite, not in figure 1 Shown in ) connection, the connection gap is sealed with wax to ensure airtightness. The syringe pump is equipped with a syringe with a volume of 10 microliters. Before use, fill the syringe with mineral oil, Teflon capillary and quartz capillary1 filled with cell lysate, and check the liquid flow path for no leaks. The small container 2 is a centrifuge tube in a U-shaped bottom 96-well plate with a maximum capacity of 200 microliters. The volume of the mineral oil 3 is 40 microliters. Droplets enclose single cells to be analyzed.

[0047] Insert the quartz capillary 1 vertically 5 mm below the liquid surface of the mineral oil 3 and keep it still. Inject 30 nanoliters of lysate with a syringe pump. After the ...

Embodiment 2

[0050] To demonstrate the precision of the microinjection employed by the present invention, we tested the reliability of using a microsyringe pump to inject 1 to 100 nanoliter volume droplets of the aqueous phase into the oil phase. Such as figure 2 , according to the microinjection filling method of Example 1, at the bottom of a small container filled with mineral oil in advance, micro-droplets with le different nanoliter level volumes were obtained. 1 is 1 nanoliter, 2 is 5 nanoliters, 3 is 10 nanoliters, 4 is 20 nanoliters, 5 is 50 nanoliters, and 6 is 100 nanoliters. Correspondingly, the flow rate of the syringe pump is 1 nL / s, 5 nL / s, 10 nL / s, 20 nL / s, 50 nL / s, 100 nL / s. Scale bar is 200 microns.

Embodiment 3

[0052] In order to demonstrate the reliability of the centrifugal microdroplet fusion adopted in the present invention, as image 3 According to the micro-droplet filling method in Example 1, two micro-droplets of 5 nanoliters were sequentially generated at the bottom of the same container. The content of droplet 1 is 0.1M FeCl 3 Solution, the content of micro-droplet 2 is 0.1M KSCN solution, two micro-droplets meet and merge to form micro-droplet 3, the contents are mixed, and the two solutions react to form reddish-brown Fe(SCN) 3 solution. Scale bar is 200 microns.

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Abstract

The invention provides a micro-volume cell nucleic acid amplification method which comprises the following steps: a) putting micro liquid droplets with a small amount of cells inside into a small-sizecontainer with an oil phase supplied in advance, and performing centrifugation to settle down the micro liquid droplets; b) putting cell lysis buffer droplets into the small-size container through micro-volume injection below the liquid level of the oil phase, performing centrifugation to settle down the cell lysis buffer droplets, and fusing the cell lysis buffer droplets with cell droplets so as to achieve splitting of cells and release of nucleic acid substances; c) adding splitting termination droplets through micro-volume injection, performing centrifugation, fusing the splitting termination droplets with the split cell droplets, and neutralizing or terminating the splitting reaction; d) adding one or more amplification reaction liquid through micro-volume injection, performing centrifugation fusion, and performing amplification on genomes, transcriptome or specific nucleotide sequences at an appropriate temperature. The micro-volume cell nucleic acid amplification method provided by the invention is simple in operation, low in cost and high in flux, and the reaction volume can be reduced to a nano liter grade.

Description

technical field [0001] The invention relates to the field of micro-liquid biochemical reaction and analysis, in particular to the technical field of cellular nucleic acid amplification based on nanoliter micro-droplet operation. Background technique [0002] Cells are the basic unit of life on earth. Nucleic acid is widely present in all animal, plant and microbial cells. Different nucleic acids have different chemical compositions and nucleotide arrangement sequences. According to different chemical composition, nucleic acid can be divided into ribonucleic acid (referred to as RNA) and deoxyribonucleic acid (abbreviated as DNA). DNA is the main material basis for storing, replicating and transmitting genetic information, while RNA plays an important role in the process of protein synthesis. Single-cell nucleic acid amplification technology is a new technology for amplifying and sequencing nucleic acid molecules such as the whole genome or transcriptome at the single-cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/686
CPCC12Q1/6844C12Q1/686C12Q2531/119C12Q2537/143C12Q2563/159
Inventor 杜文斌徐鹏贠娟莉戴欣郑小伟黄力
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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