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Nucleotide monomer capable of improving Tm value of oligonucleotide chain as well as preparation method and application thereof

A technology of nucleotide monomers and oligonucleotides, applied in the field of nucleotide monomers, can solve the problems of single structure, not high flexibility, MGB probes are not suitable for multiple fluorescence quantitative detection, etc., and achieve flexible modification methods. , the effect of improving specificity

Inactive Publication Date: 2011-10-19
李辉 +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The current MGB probe is that the MGB structure is connected to the quencher group at the 3' end, and the single structure uses DPI3. Therefore, the current MGB probe is not suitable for multiple fluorescence quantitative detection, and it is flexible for different gene designs. The performance is not high enough, resulting in the limitation of application in many places

Method used

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  • Nucleotide monomer capable of improving Tm value of oligonucleotide chain as well as preparation method and application thereof
  • Nucleotide monomer capable of improving Tm value of oligonucleotide chain as well as preparation method and application thereof
  • Nucleotide monomer capable of improving Tm value of oligonucleotide chain as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the preparation of DNA minor groove binder DQ

[0043] 1. Synthesis and purification of MGB modified dT:

[0044] Dissolve 5 mg (8.02 μmol) of DPI2 (see compound 2 in the following reaction formula, purchased from Shanghai Huirui Biotechnology Co., Ltd.) in 3ml DMF (dimethylformamide) and 3mL 0.1M NaHCO3, add 3.2mg ( 4.58 μmol) amino-dT-C6 (see compound 1 in the following reaction formula, purchased from Glen Research) was reacted at 25°C for 2 hours, desalted, acidified (60% CF3COOH-H20, 25°C, 30min), and analyzed by HPLC ( Purification by high performance liquid chromatography) gave 5.1 mg (92%) of compound 3. The specific reaction formula is as follows:

[0045]

[0046] 2. Phosphoramidation reaction:

[0047] Compound 3 (1.835g, 1.5mmol) was repeatedly rotary evaporated with anhydrous pyridine (1mL×3), dry toluene ((1mL×1) and dry acetonitrile (1mL×1) to obtain dry compound 3, which was dissolved in dry dichloromethane (15mL), bis(diisopropylami...

Embodiment 2

[0050] Example 2, the preparation of DQ (synthesis and purification of MGB modified dA):

[0051] The synthesis and purification of MGB-modified dA and the phosphoramidation reaction can be carried out according to the process of Example 1, using amino-dA-C6 (purchased from Glen Research), other substances and their molar ratios are the same as in Example 1, and the obtained compound 5 The structure is:

[0052]

[0053] The structural data of compound 5 are as follows: 1H NMR (CDC13) δ12.00 (d, 1H), 11.54 (s, 1H), 8.28 (d, 1H), 8.16 (d, 1H), 8.03 (m, 2H), 7.97 (d, 1H), 7.11-7.37(m, 12H), 6.99(m, 2H), 6.94(d, 1H), 6.81(d, 4H,), 6.37(d, 1H,), 6.21(t, 1H ,), 6.01(s, 2H), 4.62(t, 2H), 4.54-4.60(m, 1H), 4.12-4.13(m, 1H), 3.98(t, 2H), 3.75(s, 6H), 3.52 (d, 1H), 3.41 (t, 3H), 3.29 (t, 2H), 2.67-2.77 (m, 1H). m / z (M+H) of 1376.59 was found to be 1477.39 by mass spectrometry.

Embodiment 3

[0054] The preparation of embodiment 3, DQ

[0055] 1. Synthesis and purification of MGB modified dT:

[0056] Dissolve 10.12 mg (12.52 μmol) of DPI3 (see compound 6 in the following reaction formula, purchased from Shanghai Huirui Biotechnology Co., Ltd.) in 8ml DMF (dimethylformamide) and 8mL 0.1M NaHCO3, add 5mg ( 7.16 μmol) amino-dT-C6 (see compound 1 in the following reaction formula, purchased from Glen Research) was reacted at 25°C for 2 hours, desalted, acidified (60% CF3COOH-H2O, 25°C, 30min), and analyzed by HPLC ( Purification by high performance liquid chromatography) yielded 78.96 mg (89%) of the compound. The specific reaction formula is as follows:

[0057]

[0058] 2. Phosphoramidation reaction:

[0059] Compound 7 (2.111g, 1.5mmol) was repeatedly rotary evaporated with anhydrous pyridine (1mL×3), dry toluene ((1mL×1) and dry acetonitrile (1mL×1) to obtain dry compound 7, which was dissolved in dry dichloromethane (15mL), bis(diisopropylamino)(2-cyanoeth...

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Abstract

The invention discloses a nucleotide monomer capable of improving a Tm value of an oligonucleotide chain. The structure of the monomer is shown in a general formula (I), wherein R1 and R2 are hydrogen atoms or hydroxyl atoms or are connected through an oxygen atom to form locked nucleic acid; a base is a purine derivative or a pyrimidine derivative, specifically is guanine, adenine, cytosine, thymine or uracil; the base is provided with a bridge, the tail end of the bridge is provided with an active group so as to be connected with a minor groove binder (MGB), the bridge is a neutral fatty chain, and the MGB is minor groove conjugate. In addition, the invention also discloses a preparation method and application of the nucleotide monomer. The MGB is connected to single nucleotide through chemical modification, and the single nucleotide subjected to modification is used for synthesis of oligonucleotide. The nucleotide monomer is capable of improving the Tm value of the oligonucleotide chain and increasing the sensitivity; and the nucleotide monomer is connected to the base of the oligonucleotide, so that the specificity of a fluorescence quantitative polymerase chain reaction (PCR) probe is improved, and the polymorphism sites of a gene can be effectively and correctly discriminated.

Description

technical field [0001] The present invention relates to a kind of nucleotide monomer, relate in particular to a kind of nucleotide monomer with MGB, apply this nucleotide monomer (DQ) with MGB to the synthetic of oligonucleotide, can Improve the Tm value of the oligonucleotide chain; in addition, the present invention also relates to the manufacturing method of the nucleotide monomer and its application in the fields of molecular biology and the like. Background technique [0002] Real-time fluorescence quantitative PCR technology achieves quantitative purposes by detecting the intensity of fluorescent signals in PCR products. This technology not only realizes the leap from qualitative to quantitative PCR, but also has stronger specificity and effectively solves PCR pollution compared with conventional PCR. It has been widely used in the fields of animal and plant genetic engineering, microbes and medicine. [0003] At present, there are several commonly used fluorescent qu...

Claims

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Application Information

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IPC IPC(8): C07H21/04C12Q1/68
Inventor 李辉金大智方维佳
Owner 李辉
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