Linker sequence and application thereof

Abstract

The invention provides a linker sequence and an application thereof. The linker sequence comprises a molecular tag composed of a plurality of bases and sequencing linkers based on sequencing platforms; the molecular tag is located at the 3' end of the sequencing linker; and the linker sequence is linked to fragmented DNA by the molecular tag. The molecular tag composed of the plurality of bases iscombined with the sequencing linkers based on different sequencing platforms, and the constructed linker sequence is connected to one end and / or two ends of the fragmented DNA in the early stage of library construction, so that the marking effect on an original DNA fragment is realized; after the sequencing linkers are connected, the DNA is directly subjected to computer sequencing, so that an original sequence is reserved, and the technical effect of tracing the original source of the DNA fragment is achieved; and repeated fragments in the read length are removed according to the molecular tag in a sequencing result analysis process, and the sequencing accuracy is improved.

Description

technical field

[0001] The invention belongs to the technical field of molecular biology, and relates to a linker sequence and its application, in particular to a linker sequence and its application in the construction of a library and the preparation of a screening kit for carriers of monogenic genetic diseases. Background technique

[0002] Carrier screening refers to the use of economical and accurate methods to screen out carriers with normal phenotypes from the population when the incidence of a certain genetic disease is high in a specific population, in order to prevent the further development of the disease in the population , to carry out risk assessment and guidance on marriage and childbearing. The term "carrier" has a broad meaning in the medical field. In the field of genetic diseases, it mainly refers to individuals who carry a disease-causing gene (heterozygous state), but are still healthy until the time of testing.

[0003] Carrier screening can provide ris...

Examples

Embodiment 1

[0067] (1) Genomic DNA extraction

[0068] The MagPure Genomic DNA Extraction Kit was used to extract DNA from 20 samples, and the extracted DNA was stored in a -20°C refrigerator for a short period of time and stored in a -80°C refrigerator for a long time;

[0069] (2) Fragmentation and end repair

[0070] The extracted DNA sample was fragmented and end-repaired by one-step reaction using WGS-IT Frag enzyme from Qiagen Company, the reagents were mixed and centrifuged, placed on an ice box, and the reaction system was prepared according to Table 1;

[0071] After mixing, put it in a PCR machine for reaction, the reaction conditions are: 4°C for 1min, 32°C for 15min, 65°C for 30min, and store at 4°C;

[0072] After the reaction is completed, the reaction product is centrifuged briefly for the next step of ligation reaction.

[0073] Table 1

[0074] Reagent Dosage dna sample 500ng 10×WGS IT Buffer 5μL 5×WGS IT Frag 10 μL pure water Ma...

Embodiment 2

[0083] Compared with Example 1, this example also includes the step of performing PCR amplification on the library. The PCR amplification system is shown in Table 3, and the conditions are: 72°C for 5 minutes, 98°C for 2 minutes, 98°C for 20s, 58°C for 30s, 72°C for 30s, 4 cycles, 72°C for 5min, 16°C storage;

[0084] After the reaction, use the magnetic bead method to purify the product, keep the supernatant at 0.8×, keep the magnetic beads for 1.2× magnetic bead purification, dissolve in 20 μL, and then detect the library concentration;

[0085] table 3

[0086] Reagent Dosage library 20 μL 2x HiFi PCR Master Mix 25 μL Primer pair 5μL

Embodiment 3

[0088] Compared with Example 2, the molecular tag part is not included in the linker sequence, and other conditions are the same as Example 2.

technical field

[0001] The invention belongs to the technical field of molecular biology, and relates to a linker sequence and its application, in particular to a linker sequence and its application in the construction of a library and the preparation of a screening kit for carriers of monogenic genetic diseases. Background technique

[0002] Carrier screening refers to the use of economical and accurate methods to screen out carriers with normal phenotypes from the population when the incidence of a certain genetic disease is high in a specific population, in order to prevent the further development of the disease in the population , to carry out risk assessment and guidance on marriage and childbearing. The term "carrier" has a broad meaning in the medical field. In the field of genetic diseases, it mainly refers to individuals who carry a disease-causing gene (heterozygous state), but are still healthy until the time of testing.

[0003] Carrier screening can provide ris...

Examples

Embodiment 1

[0067] (1) Genomic DNA extraction

[0068] The MagPure Genomic DNA Extraction Kit was used to extract DNA from 20 samples, and the extracted DNA was stored in a -20°C refrigerator for a short period of time and stored in a -80°C refrigerator for a long time;

[0069] (2) Fragmentation and end repair

[0070] The extracted DNA sample was fragmented and end-repaired by one-step reaction using WGS-IT Frag enzyme from Qiagen Company, the reagents were mixed and centrifuged, placed on an ice box, and the reaction system was prepared according to Table 1;

[0071] After mixing, put it in a PCR machine for reaction, the reaction conditions are: 4°C for 1min, 32°C for 15min, 65°C for 30min, and store at 4°C;

[0072] After the reaction is completed, the reaction product is centrifuged briefly for the next step of ligation reaction.

[0073] Table 1

[0074] Reagent Dosage dna sample 500ng 10×WGS IT Buffer 5μL 5×WGS IT Frag 10 μL pure water Ma...

Embodiment 2

[0083] Compared with Example 1, this example also includes the step of performing PCR amplification on the library. The PCR amplification system is shown in Table 3, and the conditions are: 72°C for 5 minutes, 98°C for 2 minutes, 98°C for 20s, 58°C for 30s, 72°C for 30s, 4 cycles, 72°C for 5min, 16°C storage;

[0084] After the reaction, use the magnetic bead method to purify the product, keep the supernatant at 0.8×, keep the magnetic beads for 1.2× magnetic bead purification, dissolve in 20 μL, and then detect the library concentration;

[0085] table 3

[0086] Reagent Dosage library 20 μL 2x HiFi PCR Master Mix 25 μL Primer pair 5μL

Embodiment 3

[0088] Compared with Example 2, the molecular tag part is not included in the linker sequence, and other conditions are the same as Example 2.

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