Method for rapidly capturing and building database

A fast and linker sequence technology, applied in the field of high-throughput sequencing, can solve the problems of time-consuming, cumbersome steps, and restricting the throughput of library construction, so as to avoid large-scale amplification, increase effective concentration, and save consumption.

Active Publication Date: 2018-09-04
IGENETECH BIOTECH (BEIJING) CO LTD
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Problems solved by technology

[0003] Although the above operation process is simpler than the process of constructing the amplicon library by the enzyme ligation method, when it is applied to the library construction of high-throughput samples, the steps are still cumbersome and time-consuming, which restricts the throughput of library construction.

Method used

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  • Method for rapidly capturing and building database
  • Method for rapidly capturing and building database
  • Method for rapidly capturing and building database

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Embodiment

[0075] The extracted genomic DNA was subjected to 3 rounds of fragmentation. After each round, the sample was placed on a shaker to mix well, and after a short centrifugation, the next round of fragmentation was performed.

[0076] Take 1ul sample for capillary electrophoresis detection. After normal interruption, the main peak of the sample detection is about 150bp-200bp.

[0077] Annealing: The reaction condition is denaturation at 95°C for 5 minutes, and then annealing. The reaction condition is to drop 0.1°C every 8s to 25°C for 90 minutes. In order to form more rolling circle complexes in the ligation reaction, the ratio of the number of DNA molecules after interruption to the number of probe molecules is designed as follows: 1:5, 1:10, 1:15, 1 :20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80 , 1:85, 1:90, 1:95, 1:100, and at the same time ensure that the total amount of DNA and probe after fragmentation is 10 pmol.

[0078]

[0079] 1. Con...

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Abstract

The invention provides a method for rapidly capturing and building a database. By designing a DNA probe with the two ends provided with partial linker sequences and under the effect of CircLigase TM II ssDNA Ligase enzyme, a probe-target sequence complex, that is, a cyclized product can be formed between the DNA probe and the target sequence which is completely complementary or partially complementary with the DNA probe. The cyclized product is subjected to index amplification under the effect of phi29 polymerase and a primer with identification, the amplified product generates a great amountof database suitable for an illumia sequencing platform under the effect of a pair of sequencing primers, compared with a traditional method for capturing and building the database, the preparation time for computer database is greatly shortened, and meanwhile, the tedious database preparation process and loss of effective database in the database preparation process are avoided.

Description

technical field [0001] The invention belongs to the field of high-throughput sequencing, and in particular relates to a method suitable for constructing an amplicon library. Background technique [0002] Amplicon targeted capture technology is a targeted capture technology based on multiple PCR reactions. It uses multiple PCR reactions to amplify multiple target region sequences at the same time to obtain amplicons, and then adopts enzyme ligation or PCR reactions. The sequencing adapter sequence is introduced into the two wings of the amplicon to obtain the amplicon library, and then the next-generation sequencing is performed to obtain the sequence information of the target region. With the development of next-generation sequencing technology, the conventional liquid-phase capture method process is divided into: sample fragmentation, library construction and target region library capture. This process takes at least 2 days from genome fragmentation, library construction t...

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806C40B50/06
CPCC12N15/1093C12Q1/6806C40B50/06C12Q2525/191C12Q2531/125C12Q2525/186
Inventor 蔡万世余越美邵谦之王瑞超屈武斌杭兴宜
Owner IGENETECH BIOTECH (BEIJING) CO LTD
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