High-throughput method based on DNA (desoxyribonucleic acid) and RNA (ribonucleic acid) gene mutation detection

Abstract

The invention discloses a high-throughput method based on DNA (desoxyribonucleic acid) and RNA (ribonucleic acid) gene mutation detection. The method comprises the steps that (1) desoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are extracted from a biological sample collected from a subject through utilizing a reagent, and a DNA library is constructed based on desoxyribonucleic acid; ribonucleic acid is reversely transcribed into double-stranded cDNA firstly, and then is constructed into an RNA library; (2) a first probe set is mixed with the DNA library, and meanwhile, a second probe set is mixed with the RNA library; (3) a complex of probes and nucleic acid is separated and obtained from mixtures obtained in step (2) by utilizing markers in the first probe set and second probe set; (4) sequencing is performed on target nucleic acid in the complex by utilizing a high-throughput technique. The method is based on NGS methodology and optimizes the NGS methodology, so that the specificity and sensitivity of detection are further improved. In addition, a DNA detection result can also be verified by detecting RNA.

Description

technical field [0001] The invention relates to gene detection, in particular to a high-throughput method based on DNA+RNA gene mutation detection. Background technique [0002] Diseases such as tumors are often accompanied by a variety of somatic mutations, which are used for biological indicators such as tumor monitoring and prognosis, or targets for targeted drugs and markers of responsiveness. Tumor gene mutation types include point mutations, insertions, deletions, gene rearrangements, copy number abnormalities and other generalized gene mutations. The current common tumor somatic cell detection scheme is DNA targeted sequencing or DNA targeted sequencing combined with fluorescence quantitative PCR (qPCR), fluorescence in situ hybridization (FISH) and other auxiliary means for fusion detection. [0003] When detecting somatic mutations, researchers currently mostly choose targeted sequencing solutions, which significantly reduces the cost of sequencing and also reduces...

AI Technical Summary

Problems solved by technology

However, currently for fusion detection in somatic cell mutations, because the breakpoints of fusion genes are generally in the intron region, the intron is generally relatively long and contains regions that are not suitable for probe design, such as simple, tandem repeat sequences, transgenic For transposons, etc., if the DNA is detected alone, the design of the probe will be more difficult and will affect the capture efficiency. At the same time, considering the low abundance of the fusion gene in the total DNA sample and the low proportion of the background genome, the detection rate will be low. or the risk of missed detection

At the same time, the requirement for sequencing depth also makes the detection cost high and analysis difficult

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