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A microvolume cell nucleic acid amplification method

A cell nucleic acid and micro-volume technology, which is used in micro-liquid biochemical reactions and analysis, and in the field of cell nucleic acid amplification based on nanoliter droplet operation, which can solve the problems of complex chip processing, unsuitable for popularization and low throughput, etc.

Active Publication Date: 2021-07-02
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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  • A microvolume cell nucleic acid amplification method
  • A microvolume cell nucleic acid amplification method
  • A microvolume cell nucleic acid amplification method

Examples

Experimental program
Comparison scheme
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 microvolume cell nucleic acid amplification method, comprising: a) microdroplets encapsulating a small amount of cells are added to a small container pre-loaded with an oil phase, and centrifuged to allow it to settle; b) cell lysate droplets pass through the oil The micro-volume injection below the phase liquid level is added into a small container, and centrifuged to make it settle and fuse with the cell droplet to achieve cell lysis and release of nucleic acid substances; c) The lysis stop liquid droplet is added through a micro-volume injection, and centrifuged to make it It fuses with the lysed cell droplet to neutralize or terminate the cleavage reaction; d) one or more amplification reaction solutions are added by micro-volume injection, and centrifuged for fusion, at an appropriate temperature, the genome, transcriptome or a specific nucleic acid sequence for amplification. The cell nucleic acid amplification method provided by the invention is simple in operation, low in cost, high in throughput, and the reaction volume can be reduced to the nanoliter level.

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 Patents(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