Probe composition, next-generation sequencing library based on probe composition and application of next-generation sequencing library

Abstract

The invention discloses a probe composition, a next-generation sequencing library based on the probe composition and application of the next-generation sequencing library, and belongs to the technical field of gene detection. The probe composition provided by the invention is used for detecting the variation of a target nucleic acid sequence relative to a non-target nucleic acid sequence, and comprises a blocking probe and a capturing probe, and the blocking probe and the capturing probe are designed according to the difference sequence of the non-target nucleic acid sequence and the target nucleic acid sequence; the difference delta G non-blocking-capture between the Gibbs free energy of the combination of the blocking probe and the non-target nucleic acid sequence and the Gibbs free energy of the combination of the capture probe and the non-target nucleic acid sequence is-9.5 to-0.5 kcal / mol; the difference delta G target capture-block between the Gibbs free energy of binding of the capture probe and the target nucleic acid sequence and the Gibbs free energy of binding of the block probe and the target nucleic acid sequence is-8.5 to-0.5 kcal / mol. The probe composition disclosed by the invention is based on a selective targeted enrichment technology, and provides a construction method of a high-throughput library for high-sensitivity detection of low-frequency gene variation, and variation as low as 0.1% in a nucleic acid sample can be detected.

Description

technical field

[0001] The invention relates to the technical field of gene detection, in particular to a probe composition, a next-generation sequencing library based on the probe composition and applications thereof. Background technique

[0002] Liquid biopsy is an in vitro diagnostic technology for sampling and analyzing non-solid biological tissues such as blood. The "fluid" in a liquid biopsy is mainly blood, but also includes stool, urine, saliva, and other bodily fluid samples. The main application scenarios of liquid biopsy are early screening, diagnosis, medication guidance, monitoring, prognosis management, and non-invasive prenatal detection, in addition to musculoskeletal system and connective tissue diseases, infectious diseases and parasitic diseases, etc. disease. According to different detection substances, liquid biopsy technology has developed several branches, among which cfDNA / ctDNA technology is a common liquid biopsy technology. Free DNA (cfDNA) is ...

Examples

Embodiment 1

[0030] Hereinafter, the present invention will be further described in detail by taking the detection of hotspot mutations in non-small cell lung cancer and the Illumina high-throughput sequencing platform as examples, and in conjunction with specific examples.

[0031] 1. Probe Design and Synthesis

[0032]Aiming at the hotspots in non-small cell lung cancer-related gene mutation sites, the design and synthesis of blocking probes and capture probes are carried out according to the principle of thermodynamics. The capture probes are labeled with biotin, and the blocking probes are not biotinylated. mark. See Table 1 and Table 2 for the mutation hotspots and corresponding probe numbers of non-small cell lung cancer-related genes involved in the examples of the present invention.

[0033] Table 1 Non-small cell lung cancer gene mutation hotspots and the serial numbers of corresponding probes

[0034]

[0035]

[0036] Table 2 Probe sequence information

[0037]

[00...

technical field

[0001] The invention relates to the technical field of gene detection, in particular to a probe composition, a next-generation sequencing library based on the probe composition and applications thereof. Background technique

[0002] Liquid biopsy is an in vitro diagnostic technology for sampling and analyzing non-solid biological tissues such as blood. The "fluid" in a liquid biopsy is mainly blood, but also includes stool, urine, saliva, and other bodily fluid samples. The main application scenarios of liquid biopsy are early screening, diagnosis, medication guidance, monitoring, prognosis management, and non-invasive prenatal detection, in addition to musculoskeletal system and connective tissue diseases, infectious diseases and parasitic diseases, etc. disease. According to different detection substances, liquid biopsy technology has developed several branches, among which cfDNA / ctDNA technology is a common liquid biopsy technology. Free DNA (cfDNA) is ...

Examples

Embodiment 1

[0030] Hereinafter, the present invention will be further described in detail by taking the detection of hotspot mutations in non-small cell lung cancer and the Illumina high-throughput sequencing platform as examples, and in conjunction with specific examples.

[0031] 1. Probe Design and Synthesis

[0032]Aiming at the hotspots in non-small cell lung cancer-related gene mutation sites, the design and synthesis of blocking probes and capture probes are carried out according to the principle of thermodynamics. The capture probes are labeled with biotin, and the blocking probes are not biotinylated. mark. See Table 1 and Table 2 for the mutation hotspots and corresponding probe numbers of non-small cell lung cancer-related genes involved in the examples of the present invention.

[0033] Table 1 Non-small cell lung cancer gene mutation hotspots and the serial numbers of corresponding probes

[0034]

[0035]

[0036] Table 2 Probe sequence information

[0037]

[00...

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