SNP composite detection system based on genome simplification and next-generation sequencing and detection method thereof

Abstract

The invention discloses an SNP composite detection system based on genome simplification and next-generation sequencing and a detection method thereof. The detection method comprises the following steps: adding substrates into formed amplification products to synthesize first bases, removing all the free bases, selecting a PTP flat plate during sequencing, wherein 1600000 optical fiber holes areincluded in the surface of the PTP flat plate, and various enzymes and substrates required by chemical luminous reaction are loaded in the optical fiber holes, then successively and circularly introducing the bases into the PTP flat plate according to a sequence of T, A, C and G, releasing pyrophosphoric acid if the bases are paired, forming oxyluciferin via the pyrophosphoric acid under the action of the enzyme, simultaneously releasing optical signals, capturing the optical signals by using a high-sensitivity CCD in real time, pairing the bases with the PTP flat plate, then capturing the optical signal of one molecule, thus realizing one-to-one correspondence to accurately and rapidly determine base sequences of templates to be detected. The system and the method are capable of accurately sequencing genomes.

Description

technical field

[0001] The invention relates to the technical field of genome sequencing, in particular to a genome simplification and next-generation sequencing SNP composite detection system and detection method. Background technique

[0002] As an important experimental technique, DNA sequencing has been widely used in biological research. As early as shortly after the discovery of the DNA double helix structure (Watson and Crick, 1953), DNA sequencing technology was reported, but the operation process at that time was complicated and could not be scaled up. The Sanger method has become the mainstream of DNA sequencing so far because it is simple and fast, and has been continuously improved. However, with the development of science, traditional Sanger sequencing can no longer fully meet the needs of research. Genome resequencing of model organisms and genome sequencing of some non-model organisms require lower cost, higher throughput, and faster speed. The second-genera...

Examples

Embodiment 1

[0027] The present invention proposes a genome simplification and next-generation sequencing SNP composite detection system and detection method, including the following steps:

[0028] S1: Select the target DNA and digest the target DNA with an endonuclease to obtain the target DNA fragment. After completion, place it in a sterile storage box at minus 10 degrees Celsius for standby;

[0029] S2: Select the DNA template again, and apply the DNA template preparation solution on the surface of the DNA template, and the DNA template preparation solution should be evenly applied to ensure the accuracy of the reaction;

[0030] S3: Add a DNA repair enzyme to the target DNA fragment processed in S1, and repair the end of the target DNA fragment under an environment of 15 degrees Celsius, then add bases to it again, store it for 3 hours, and then use it;

[0031] S4: Take out the target DNA fragments stored in S3, place them in a reaction vessel at 15 degrees Celsius, and connect the...

Embodiment 2

[0048] The present invention proposes a genome simplification and next-generation sequencing SNP composite detection system and detection method, including the following steps:

[0049] S1: Select the target DNA and digest the target DNA with an endonuclease to obtain the target DNA fragment. After completion, place it in a sterile storage box at minus 8 degrees Celsius for standby;

[0050] S2: Select the DNA template again, and apply the DNA template preparation solution on the surface of the DNA template, and the DNA template preparation solution should be evenly applied to ensure the accuracy of the reaction;

[0051]S3: Add a DNA repair enzyme to the target DNA fragment processed in S1, and repair the end of the target DNA fragment under an environment of 18 degrees Celsius, and then add bases to it again, store it for 4 hours, and then use it;

[0052] S4: Take out the target DNA fragments stored in S3, place them in a reaction vessel at 18 degrees Celsius, and connect t...

Embodiment 3

[0069] The present invention proposes a genome simplification and next-generation sequencing SNP composite detection system and detection method, including the following steps:

[0070] S1: Select the target DNA and digest the target DNA with an endonuclease to obtain the target DNA fragment. After completion, place it in a sterile storage box at minus 6 degrees Celsius for use;

[0071] S2: Select the DNA template again, and apply the DNA template preparation solution on the surface of the DNA template, and the DNA template preparation solution should be evenly applied to ensure the accuracy of the reaction;

[0072] S3: Add a DNA repair enzyme to the target DNA fragment processed in S1, and repair the end of the target DNA fragment at 22 degrees Celsius, then add bases to it again, store it for 5 hours, and then use it;

[0073] S4: Take out the target DNA fragments stored in S3, place them in a reaction vessel at 22 degrees Celsius, and connect the target DNA fragments with...

technical field

[0001] The invention relates to the technical field of genome sequencing, in particular to a genome simplification and next-generation sequencing SNP composite detection system and detection method. Background technique

[0002] As an important experimental technique, DNA sequencing has been widely used in biological research. As early as shortly after the discovery of the DNA double helix structure (Watson and Crick, 1953), DNA sequencing technology was reported, but the operation process at that time was complicated and could not be scaled up. The Sanger method has become the mainstream of DNA sequencing so far because it is simple and fast, and has been continuously improved. However, with the development of science, traditional Sanger sequencing can no longer fully meet the needs of research. Genome resequencing of model organisms and genome sequencing of some non-model organisms require lower cost, higher throughput, and faster speed. The second-genera...

Examples

Embodiment 1

[0027] The present invention proposes a genome simplification and next-generation sequencing SNP composite detection system and detection method, including the following steps:

[0028] S1: Select the target DNA and digest the target DNA with an endonuclease to obtain the target DNA fragment. After completion, place it in a sterile storage box at minus 10 degrees Celsius for standby;

[0029] S2: Select the DNA template again, and apply the DNA template preparation solution on the surface of the DNA template, and the DNA template preparation solution should be evenly applied to ensure the accuracy of the reaction;

[0030] S3: Add a DNA repair enzyme to the target DNA fragment processed in S1, and repair the end of the target DNA fragment under an environment of 15 degrees Celsius, then add bases to it again, store it for 3 hours, and then use it;

[0031] S4: Take out the target DNA fragments stored in S3, place them in a reaction vessel at 15 degrees Celsius, and connect the...

Embodiment 2

[0048] The present invention proposes a genome simplification and next-generation sequencing SNP composite detection system and detection method, including the following steps:

[0049] S1: Select the target DNA and digest the target DNA with an endonuclease to obtain the target DNA fragment. After completion, place it in a sterile storage box at minus 8 degrees Celsius for standby;

[0050] S2: Select the DNA template again, and apply the DNA template preparation solution on the surface of the DNA template, and the DNA template preparation solution should be evenly applied to ensure the accuracy of the reaction;

[0051]S3: Add a DNA repair enzyme to the target DNA fragment processed in S1, and repair the end of the target DNA fragment under an environment of 18 degrees Celsius, and then add bases to it again, store it for 4 hours, and then use it;

[0052] S4: Take out the target DNA fragments stored in S3, place them in a reaction vessel at 18 degrees Celsius, and connect t...

Embodiment 3

[0069] The present invention proposes a genome simplification and next-generation sequencing SNP composite detection system and detection method, including the following steps:

[0070] S1: Select the target DNA and digest the target DNA with an endonuclease to obtain the target DNA fragment. After completion, place it in a sterile storage box at minus 6 degrees Celsius for use;

[0071] S2: Select the DNA template again, and apply the DNA template preparation solution on the surface of the DNA template, and the DNA template preparation solution should be evenly applied to ensure the accuracy of the reaction;

[0072] S3: Add a DNA repair enzyme to the target DNA fragment processed in S1, and repair the end of the target DNA fragment at 22 degrees Celsius, then add bases to it again, store it for 5 hours, and then use it;

[0073] S4: Take out the target DNA fragments stored in S3, place them in a reaction vessel at 22 degrees Celsius, and connect the target DNA fragments with...