High-flux single-cell whole genome amplification method

Abstract

The invention discloses a high-flux single-cell whole genome amplification method. The method comprises the steps as follows: a cell suspension with preset cell density is distributed to each micropore of a nano microporous chip by use of a nano micro liquid distribution platform; a cell lysis buffer is distributed to micropores by use to the micro liquid distribution platform to lyse cells in the micropores; a neutralization solution is distributed to micropores by use to the micro liquid distribution platform for a neutralization reaction; a whole genome amplification reagent is distributed to micropores by use to the micro liquid distribution platform for a whole genome amplification reaction. The method has the characteristics of being high in flux, low in cost, single-cell and integrated, and automatic and large-scale single-cell whole genome amplification can be realized.

Description

technical field [0001] The invention relates to the technical field of single-cell whole-genome amplification, in particular to a high-throughput single-cell whole-genome amplification method. Background technique [0002] Studies have shown that in the process of embryonic development, disease occurrence, and tumor development, due to the individual differences and heterogeneity of the genetic material of a single cell, it leads to extremely important or even decisive consequences. Therefore, it is necessary to sequence the genome of a single cell of. At present, single-cell genome sequencing has been successfully applied to various microbial and mammalian cells. [0003] In single-cell research work, scaling up experiments is key to ensuring that sufficient biodiversity information is collected. Although single cells can be separated and samples can be amplified using microfluidic chips, the throughput is not high. Other methods for single cells have many defects, resul...

AI Technical Summary

Problems solved by technology

Although single cells can be separated and samples can be amplified using microfluidic chips, the throughput is not high

Other methods for single cells have many defects, resulting in low detection sensitivity, serious loss of gene expression information, high technical noise, high operation error rate, and poor repeatability. The cost has become the biggest bottleneck again. The high cost of single cell expansion leads to the immature stage of single cell expansion, and it is far from reaching the point of large-scale application in terms of technology and cost.

[0004] At present, the most common and simplest method for amplifying the genomic DNA of a single cell is the multiple displacement amplification (MDA) technique, which uses random primers and isothermal amplification to obtain a large number of high-fidelity DNA fragments, but This method has problems such as amplification bias and non-specific amplification.

In addition, other developed methods such as the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) cannot detect single nucleotide variations due to their low gene coverage

Although the multiple annealing and looping-based amplification cycles (MALBAC) method can suppress amplification bias, its fidelity is poor and the operation steps are relatively complicated, which is not conducive to high-throughput whole-genome amplification. (WGA)

[0005] At present, commercial single-cell WGA kits are not only expensive, but the reagent components are kept confidential, and the reaction volume is around tens of microliters (μL), and the amplification products are biased.

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