Homocysteine aptamer HCy2 and preparation method thereof

A homocysteine, cystine nucleic acid technology, applied in the field of amino acid detection, can solve the problems of lack of efficient and specific identification of homocysteine, screening preparation methods have not yet been reported, etc., and achieves easy synthesis and labeling. , Beneficial to design, improving specificity and sensitivity

Inactive Publication Date: 2015-04-29
陈燕婷 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of efficient and specific recognition of homocysteine ​​based on nucleic acid aptamers,

Method used

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  • Homocysteine aptamer HCy2 and preparation method thereof
  • Homocysteine aptamer HCy2 and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Example

[0028] Implementation 1 Preparation of aptamer HCy2 that specifically binds homocysteine

[0029] Construction of random sequence oligonucleotide library: artificially synthesize single-stranded DNA sequences with a library capacity of 1×10 5 Single-stranded DNA random sequence oligonucleotide library, the DNA sequence included in the single-stranded DNA random sequence oligonucleotide library is:

[0030] 5’-GGATCCACCAGCGTCATCAGCA-N 25~40 -AGATAGTAAGTGCAATCTGGC-3’

[0031] The DNA sequence of the single-stranded DNA random oligonucleotide library includes an intermediate random sequence N 25~60 And fixed sequence at both ends, the middle random sequence N 25~40 It is a random sequence of 30-40 bases, the fixed sequence at both ends is: 5’-GGATCCACCAGCGTCATCAGCA, 3’-CGGTCTAACGTGAATGATAGA, and the fixed sequence at both ends is the PCR amplification primer binding region

[0032] 1) Put 0.5ml (1x10 9 Microparticles) Invitrogen's micromagnetic beads with activated amino groups and 1m...

Example Embodiment

[0040] Example 2 Detecting the binding ability of aptamer HCy2 and homocysteine ​​by flow cytometry

[0041] 1) Synthesize a homocysteine ​​aptamer labeled with a fluorescent group FAM at the 5'end.

[0042] 2) Use 0nml / L, 5nml / L, 10nml / L, 20nml / L, 50nml / L, 100nml / L, 200nml / L concentration gradient FAM-labeled nucleic acid aptamer to connect homocysteine ​​(HCy) micro Magnetic beads to determine the dissociation constant (kd) of homocysteine ​​aptamers. The above-mentioned nucleic acid aptamers of various concentrations diluted with 200 μL of binding buffer were added with 150 nmol / L homocysteine-linked micromagnetic beads, and incubated at 37°C for 30 minutes. After washing the magnetic beads with binding buffer, resuspend them in 250 μL of binding buffer. Set random sequence oligonucleotide fragments and cysteine ​​(Cy)-linked micromagnetic beads as controls.

[0043] 3) Use BD's flow cytometer to measure the fluorescence of the beads, and then use the Sigma plot software to plo...

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Abstract

The invention discloses a homocysteine aptamer with high specificity and high affinity and a preparation method of the homocysteine aptamer. The homocysteine aptamer has the nucleotide sequence as follows: gggaggcatg agcattagtg cgcaacagct ctggtactgg attggaaccc and is named as HCy2. The preparation method of the homocysteine aptamer comprises the steps of establishing a random sequence library; screening a target substance and nucleic acid complex; separating a target oligonucleotide aptamer; carrying out PCR amplification on the target oligonucleotide aptamer; circularly screening a high-specificity aptamer; and modifying the homocysteine aptamer. The prepared aptamer is nontoxic, small in molecular weight, easy to synthesize and mark, only capable of specifically recognizing homocysteine, and incapable of recognizing and combining other homocysteines or analogues of the other homocysteines so as to become a molecular beacon for detecting the content of the homocysteine.

Description

technical field [0001] The invention belongs to the technical field of amino acid detection and relates to a nucleic acid, in particular to a homocysteine ​​nucleic acid aptamer with high specificity and high affinity and a preparation method thereof. Background technique [0002] Homocysteine ​​(HCys), also known as homocysteine ​​or homocysteine, is a heterogeneous amino acid cysteine, which contains a thiol group (-SH) in the side chain Additional methylene (-CH2-). Homocysteine ​​is a risk factor for many diseases. At present, the increase of homocysteine ​​is considered to be the most extensive and independent pathogenic factor of cardiovascular and cerebrovascular diseases caused by atherosclerosis. Compared with the traditional three blood lipids, cholesterol, high-density protein, and triglyceride, homocysteine ​​can be used as an independent indicator for judging the risk of cardiovascular and cerebrovascular diseases. Since the increase of homocysteine ​​is gradua...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12N15/10C07K14/00G01N15/14
Inventor 陈燕婷张莘蔓李红梅
Owner 陈燕婷
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