Nucleic acid aptamer screening method

A screening method and nucleic acid aptamer technology, which can be used in biochemical equipment and methods, and the determination/inspection of microorganisms, and can solve problems such as no candidate nucleic acid aptamer screening methods.

Pending Publication Date: 2021-11-26
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There is no sequencing-based candidate nucleic acid aptamer screening method in the prior art

Method used

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  • Nucleic acid aptamer screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] To the nucleic acid sample to promote nucleic acid samples to form a secondary structure, amplification and sequencing of nucleic acid samples, including the following steps:

[0033] (1) The genomic DNA was extracted from phenol-chloroform from the cultured human K562 cell line and dissolved in TE buffer. The nucleic acid sample to be tested is divided into two groups, each containing 10 μl of a total of 100 ng of genome nucleic acid.

[0034] (2) Add a final concentration of 1 μm MnCl to the above-mentioned nucleic acid sample 2 The solution enables both to mix; another group of isometric water.

[0035] (3) The MDA total genome is added to the MDA total genomic, 4 mmDNTP, 1U / μl29 polymerase, and corresponding buffer, respectively, the reaction conditions for the MDA total genome, and the reaction conditions were carried out on a 50 μm random primer, 4 mmdNTP, 1 U / μL29 polymerase and corresponding buffer.

[0036] (4) After the amplification, the two sets of samples wer...

Embodiment 2

[0040] According to the method of Example 1, a sequence of artificially synthetic nucleic acids were screened by a sequence of nucleic acids.

[0041]

[0042] 2 μl of concentration of 100 μm Pb (NO) was added to a nucleic acid sample of 198 μL of concentration of 20 μm (NO 3 ) 2 The solution allows it to fully react. Another group of sample nucleic acid concentrations are maintained, but add the same molar NANO 3 Solution.

[0043] The amplification of the primary ionic nucleic acid sample set and the non-lead ion-free nucleic acid sample group was amplified, and the PCR (polymerase chain reaction) was amplified. The PCR reaction system contains 400 μm DNTPs, 2 μm primers, 0.025 unit / μl RTAQ and 1 μL of the template in 50 μl of 1 × PCR buffer. The process of PCR is denaturation (92 ° C, 15S), annealing (58 ° C, 30S), extending (72 ° C, 20S), a total of 14 cycles, and finally extends at 72 ° C for 5 min.

[0044] After amplification, the two groups of samples were subjected to...

Embodiment 3

[0047] According to the method of Example 1, a PDS nucleic acid aptamer was performed on a natural nucleic acid sequence from human lymphocytes. The final concentration of 1 μm PDS was added to a nucleic acid sample having a concentration of 20 μm to sufficiently reacted. Another group of sample nucleic acid concentrations is maintained, but is added to the same amount of water.

[0048] The amplification of the PDS-containing nucleic acid sample group and the Nucleic acid sample group containing the PDS were amplified by PCR (polymerase chain reaction). The PCR reaction system contains 400 μm DNTPs, 2 μm primers, 0.025 unit / μl RTAQ and 1 μL of the template in 50 μl of 1 × PCR buffer. The process of PCR is denaturation (98 ° C, 15S), annealing (58 ° C, 30S), extending (72 ° C, 90S), a total of 14 cycles, and finally extends at 72 ° C for 5 min.

[0049] After amplification, the two groups of samples were subjected to ultrasound interruption and ligation enzyme connection sequenc...

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PUM

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Abstract

The invention discloses a nucleic acid aptamer screening method. The nucleic acid aptamer screening method comprises the following steps: dividing nucleic acid samples to be detected into two groups, amplifying the first group of nucleic acid samples under the condition of not containing a target substance or not amplifying the first group of nucleic acid samples, and amplifying the second group of nucleic acid samples under the condition of containing the target substance; respectively sequencing the two groups of amplified nucleic acid samples; and after sequencing is completed, comparing coverage depth differences of the nucleic acid sequences in the two groups of nucleic acid samples, finding out the nucleic acid sequences with obviously reduced coverage depth in the second group, and screening out candidate nucleic acid aptamers. The screening method can more simply, conveniently and quickly screen the nucleic acid aptamers on a large scale, and is suitable for screening candidate nucleic acid aptamers from long-sequence nucleic acid and multi-species genomes.

Description

Technical field [0001] The present invention relates to biotechnology, and more particularly to a nucleic acid aptamer screening method. Background technique [0002] Nucleic acid antibody is generally a single-stranded oligonucleotide sequence having a molecular size of 25 to 60 nucleotides, which produces a specific molecular interaction between three-dimensional structures and target molecules by folding into a stable three-dimensional structure, which can specifically bind proteins or Other small molecular substances. Nucleic acid antibody has a specificity and affinity of antibody, and has a wide range of targets, low production costs, and easy-to-scale preparation, which can achieve precision chemical synthesis and modification and low immunogenicity. [0003] Currently use of a wide range of nucleic acid split screens is the use of Index Evolution Ofligands By Exponential Enrichment, SELEX, screening from random single-stranded nucleic acid sequence libraries, specificity ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6811
CPCC12Q1/6811C12Q2525/205C12Q2537/165
Inventor 涂景李王玥乔祎罗雨菡
Owner SOUTHEAST UNIV
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