Method for high-throughput detection of gene mutation in single cells at full-length transcript level

Abstract

The invention discloses a method for high-throughput detection of gene mutation in single cells at a full-length transcript level and relates to the field of nucleic acid detection. The method comprises the following steps of: marking the single cells by utilizing a single cell analyzer and marking various mRNA (messenger Ribonucleic Acid) in the single cells; amplifying and utilizing a specific primer of a gene to be detected to amplify and enrich a full-length cDNA (complementary Deoxyribonucleic Acid) sequence of the gene; constructing a three-generation sequencing library through a segmentobtained by amplification and sequencing a full-length cDNA library of the gene to be detected through a PacBio sequencer, so as to obtain sequence information of the gene to be detected at a singlecell level.

Description

Technical field [0001] The present invention relates to the field of nucleic acid detection, in particular to a method for high-throughput detection of gene mutations in single cells at the full-length transcript level. Background technique [0002] The mutation detection of gene transcripts refers to the technique of using experimental methods to identify the mutation sites produced by the gene sequence of the RNA expressed by an individual. [0003] The detection of mutations can be done at the genome level or at the transcriptome level. If the genome is used as a sample to detect and identify individual sequence mutations, DNA extraction can be used to amplify the target sequence with specific primers and then perform first-generation sequencing to identify mutations. The extracted DNA can also be used to construct a second-generation sequencing library. After the library is sequenced, the sequencing data obtained can be analyzed to obtain information about individual gene muta...

AI Technical Summary

Problems solved by technology

The throughput of the former technique is low, but it is more economical and fast, and it is still a common method for identifying sequence mutations.

The second method has the advantages of large amount of data and high throughput, and the sequence mutation information in most genomes can be obtained through one-time sequencing, but the disadvantage is that the cycle is long, the analysis complexity is high, and the cost is high.

However, because most of the current single-cell analyzers use the next-generation sequencing method for sequence determination, the sequencing length is limited, and some long-fragment sequences cannot be spliced ​​after sequencing, resulting in the inability to obtain mutation information of transcripts. Therefore, when identifying full-length transcripts This mutation is subject to certain restrictions, and some research work cannot be completed