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Quick identification and separation method of affine nucleic acid molecule

A nucleic acid molecule and molecular technology, applied in the field of identification and separation, can solve the problems of different sequence amplification efficiency, high cost, and inability to quickly and directly obtain a single sequence nucleic acid molecule, and achieve the effect of avoiding false positives

Active Publication Date: 2014-01-29
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is costly and requires high-throughput sequencing instruments, and only a few laboratories can adopt this method; although this method does not require cloning, it can only obtain sequence information, and still needs to pass synthesis and affinity verification before it can be used. get a specific molecule
Moreover, due to unequal amplification and different amplification efficiencies of different sequences, high-abundance sequences may not necessarily be high-affinity sequences
Therefore, none of the above existing methods can quickly and directly obtain high-affinity single-sequence nucleic acid molecules

Method used

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  • Quick identification and separation method of affine nucleic acid molecule

Examples

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Embodiment 1

[0044] Example 1: Rapid identification of single-stranded DNA nucleic acid aptamers

[0045] This embodiment is a preferred embodiment, specifically as follows:

[0046] 1. Coupling of primers on the surface of microbeads; either covalent coupling or non-covalent coupling of primers to microbeads;

[0047] Take 50uL polystyrene (PS) microbeads with carboxyl groups (as microbeads A), wash twice with 0.01M NaOH solution (the specific method is: centrifuge the sample at 8000rpm for 12min, discard the supernatant, add 0.01M NaOH Solution, mix well and let stand for 5min, then centrifuge again), wash with deionized water 3 times, discard the supernatant; dissolve the amino-modified primer with deionized water, the final concentration is 100uM, take out 20uL, add In the centrifuge tube of ethylene microbeads, add the same amount of deionized water to the control group; add 50uL MES buffer solution (0.4M MES, pH5.0) into the tube; shake at 300 rpm, shake and mix for 30min; add 30uL...

Embodiment 2

[0070] Example 2: Promoter Screening

[0071] If you need to screen double-stranded DNA sequences that bind to transcription factors, you can follow the same process as above: first construct a library containing various possible promoter recognition sequences, and use emulsion PCR to amplify these sequences into different microbeads A Above, refer to steps 1, 2, 3, 5, and 6 in Example 1 to carry out. Since the double-stranded DNA sequence is screened here, there is no need to melt the PCR product on microbead A. If specific sequence information is required, it is only necessary to sequence the sequence on a single bead.

Embodiment 3

[0072] Example 3: Rapid Identification of RNA Molecules

[0073] If it is necessary to identify the binding of the RNA molecule library to the target, the steps in Example 1 can be referred to, but the following improvements need to be made:

[0074] Due to the difficulty of directly utilizing the RNA-dependent RNA polymerase to amplify the RNA molecule, the following scheme is adopted in the present invention:

[0075] First, couple the two sequences on microbead A at a molar ratio of 1:100, as described in Step 1 of Example 1; among the two coupled sequences, the one with the smaller amount is a primer for amplifying DNA Sequences, the one with a large number is the paired sequence used to capture RNA molecules, where the end of the paired sequence used to capture RNA molecules is blocked and modified so that it cannot be used for extension and amplification;

[0076] Secondly, the RNA molecular library was reverse-transcribed into DNA, operated according to the instruction...

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Abstract

The invention discloses a method for quickly obtaining molecules with characteristics from a complicated nucleic acid library. The method comprises the following steps: firstly, respectively amplifying a single molecule of the library on a single micro-bead A by an emulsion nucleic acid amplification technique, so that only a nucleic acid molecule with a specific sequence is formed on each micro-bead A; coupling a target molecule on another micro-bead B (such as a magnetic bead) with different properties, mixing the two micro-beads together to combine, and then cleaning uncombined micro-bead A, and separating the micro-bead B with the target molecule, so that the micro-bead A combined with the target molecule is separated together; the nucleic acid molecule which is specifically combined with the target molecule on the micro-bead B on the micro-bead A is a required single sequence nucleic acid molecule. The method is fast and convenient; the single nucleic acid molecule with high affinity can be obtained without cloning and sequencing. The technique can be widely applied to screening of promoters, and screening and identifying of aptamers.

Description

technical field [0001] The invention relates to an identification and separation method, in particular to a method for quickly identifying and isolating molecules with certain characteristics from a mixed nucleic acid molecule library. Background technique [0002] Nucleic acid molecules, including DNA and RNA, play an extremely important role in living organisms. Some of the nucleic acid molecules can interact with other molecules, and through this interaction, information can be transmitted and specific functions can be performed. Such nucleic acid molecules capable of interacting with other molecules are not only ubiquitous in living organisms, but also can be screened in vitro to obtain nucleic acid molecules with specific functions. This in vitro screening technology is called SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology, which was independently invented by three laboratories in the United States in 1990. [0003] Whether it is looking ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/115C12N15/10
CPCC12Q1/686C12Q2563/149C12Q2563/159
Inventor 罗昭锋罗炎欧惠超周宏敏张海燕
Owner UNIV OF SCI & TECH OF CHINA
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