High-throughput sequencing library construction method of nucleic acid aptamer library

A nucleic acid aptamer, sequencing library technology, applied in chemical libraries, DNA preparation, combinatorial chemistry, etc., can solve the problems of PCR amplification preference, primer residues, etc., to avoid base preference, increase output and analysis, To achieve the effect of complete retention

Inactive Publication Date: 2021-01-15
BIOMARKER TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In order to achieve the above purpose, the present invention removes the residual primers in the nucleic acid aptamer library due to SELEX amplification before the construction of the sequencing library, and then uses the PCR-Free technology to construct the sequencing library, effectively solving the problem of PCR amplification preference and The problem of primer residues, more accurately retains the original information of the nucleic acid aptamer library, and improves the accuracy of aptamer library screening

Method used

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  • High-throughput sequencing library construction method of nucleic acid aptamer library
  • High-throughput sequencing library construction method of nucleic acid aptamer library
  • High-throughput sequencing library construction method of nucleic acid aptamer library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 High-throughput sequencing library construction method of nucleic acid aptamer library (1)

[0048] This embodiment provides a method for constructing a high-throughput sequencing library of a nucleic acid aptamer library. The schematic flow chart of the method is as follows: figure 1 As shown, it specifically includes the following steps:

[0049] 1. Use the QIAGEN MinElute PCR Purification Kit to purify the nucleic acid aptamer PCR product mixture library

[0050] (1) Take 5 times the volume of PB of the sample to be purified and mix it with the sample to be purified, add it to the silica gel membrane purification column, place the silica gel membrane purification column on a 2mL collection tube, and let it stand for 2 minutes, so that the nucleic acid and the column membrane are fully combined.

[0051] (2) Centrifuge at 8000rpm for 1min, and discard the waste liquid.

[0052] (3) Add 750 μL PE (absolute ethanol has been added and mix well) to the purifi...

Embodiment 2

[0066] Example 2 High-throughput sequencing library construction method of nucleic acid aptamer library (2)

[0067] This example provides a method for constructing a high-throughput sequencing library of a nucleic acid aptamer library, which differs from the method in Example 1 only in that in step 3, the reaction is performed at 24°C for 10 minutes and at 22°C for 20 minutes.

experiment example 2

[0072] Analysis of the quality output and sequencing data output of the sequencing library in Experimental Example 2

[0073] The quality output of the library and the output analysis of the sequencing data were carried out on the high-throughput sequencing library constructed by the library construction method of the above Example 1, Example 2 and Comparative Example 1. The results are shown in Table 1 and Table 2. The results show that , the sequencing library of the nucleic acid aptamer library sample constructed by the method of Example 1 has a higher concentration and about 90% of effective data, and the sequencing library of the nucleic acid aptamer library sample constructed by the method of Example 2 is higher than that of the implementation The concentration and effective data of the library in Example 1 are slightly inferior, and the effective data of sequencing can be about 80%. The effective concentration, QPCR concentration and data output of the library S059-R01 c...

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Abstract

The invention relates to the technical field of nucleic acid aptamer screening, in particular to a high-throughput sequencing library construction method of a nucleic acid aptamer library. The sequencing library construction method provided by the invention comprises the following steps of removing primer residues from an aptamer library obtained by targeted screening, and then constructing a PCR-Free sequencing library. According to the sequencing library construction method disclosed by the invention, primer residues existing in the nucleic acid aptamer library are reduced by utilizing a silica gel column membrane purification technology, so that the output and analysis of subsequent effective data are increased, and complete and accurate reservation of information of the nucleic acid aptamer library is realized; the PCR-Free library construction technology is used for avoiding aerosol pollution in the PCR amplification process and base preference caused by PCR amplification preference and amplification enzyme performance difference, the effectiveness of sequencing data analysis is guaranteed, and meanwhile the construction efficiency and quality of a sequencing library are guaranteed.

Description

technical field [0001] The invention relates to the technical field of nucleic acid aptamer screening, in particular to a high-throughput sequencing library construction method for a nucleic acid aptamer library. Background technique [0002] Nucleic acid aptamer is a short single-stranded DNA / RNA that can bind to the target with high affinity and specificity. It not only has a highly stable three-dimensional structure and low immunogenicity, but also is easy to synthesize and has no difference between batches. Widely used in environmental monitoring, food safety inspection and clinical diagnosis and treatment. The nucleic acid aptamer library can be enriched from different types of ligands by exponential enrichment system evolution technology (systematic evolution of ligands by exponential Enrichment, SELEX) from 10 14 ~10 16 These single-stranded nucleic acid sequences consist of random sequences in the middle and fixed primer binding sequences at both ends. [0003] Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12N15/10
CPCC40B50/06C12N15/1034
Inventor 郑洪坤骆晨张雪川许国路李绪明康靖民毕经德
Owner BIOMARKER TECH
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