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Method for reducing fluorescent background of Taqman probe

A fluorescent background and probe technology, applied in the field of DNA synthesis, can solve the problem of difficult separation of Taqman probes, and achieve the effect of avoiding the existence and reducing the fluorescent background.

Pending Publication Date: 2021-06-18
通用生物(安徽)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, it is difficult for chemical products to be 100% pure. Therefore, there will be a small amount of unlabeled CPG in the 3-terminal quenching group CPG raw material of Taqman probe. Protection, coupling, capping, and oxidation four-step cycle reaction, the obtained Taqman probe will have a small amount of fluorescently labeled by-products that are not easy to separate during the purification process, showing a slightly higher background fluorescence in the subsequent qPCR detection

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Embodiment 1

[0023] A method for reducing the fluorescent background of Taqman probes, specifically comprising the steps of:

[0024] Step A1: Prepare the Taqman probe, the probe sequence is 5'FAM-TTGCTGCTGCTTGACAGATT-BHQ13', fill the Taqman probe into the synthesis column, put the synthesis column into the synthesis plate, add acetic anhydride and 1-methylimidazole, and block After the CPG without quenching group is added, trichloroacetic acid is added for reaction, and the protecting group DMT is removed to obtain a free 5′ hydroxyl solution;

[0025] Step A2: Mix the phosphoramidite-protected nucleotide monomer with the activator tetrazolium to prepare the nucleoside phosphorous acid activated intermediate, and perform condensation reaction between the nucleoside phosphorous acid activated intermediate and the free 5′ hydroxyl solution , add acetic anhydride and 1-methylimidazole, after terminating the reaction, add iodine, carry out oxidation reaction, make the deoxyribonucleotides be ...

Embodiment 2

[0030] A method for reducing the fluorescent background of Taqman probes, specifically comprising the steps of:

[0031] Step A1: Prepare the Taqman probe, the probe sequence is 5'FAM-TTGCTGCTGCTTGACAGATT-BHQ13', fill the Taqman probe into the synthesis column, put the synthesis column into the synthesis plate, add acetic anhydride and 1-methylimidazole, and block After the CPG without quenching group is added, trichloroacetic acid is added for reaction, and the protecting group DMT is removed to obtain a free 5′ hydroxyl solution;

[0032] Step A2: Mix the phosphoramidite-protected nucleotide monomer with the activator tetrazolium to prepare the nucleoside phosphorous acid activated intermediate, and perform condensation reaction between the nucleoside phosphorous acid activated intermediate and the free 5′ hydroxyl solution , add acetic anhydride and 1-methylimidazole, after terminating the reaction, add iodine, carry out oxidation reaction, make the deoxyribonucleotides be ...

Embodiment 3

[0037] A method for reducing the fluorescent background of Taqman probes, specifically comprising the steps of:

[0038]Step A1: Prepare the Taqman probe, the probe sequence is 5'FAM-TTGCTGCTGCTTGACAGATT-BHQ13', fill the Taqman probe into the synthesis column, put the synthesis column into the synthesis plate, add acetic anhydride and 1-methylimidazole, and block After the CPG without quenching group is added, trichloroacetic acid is added for reaction, and the protecting group DMT is removed to obtain a free 5′ hydroxyl solution;

[0039] Step A2: Mix the phosphoramidite-protected nucleotide monomer with the activator tetrazolium to prepare the nucleoside phosphorous acid activated intermediate, and perform condensation reaction between the nucleoside phosphorous acid activated intermediate and the free 5′ hydroxyl solution , add acetic anhydride and 1-methylimidazole, after terminating the reaction, add iodine, carry out oxidation reaction, make the deoxyribonucleotides be c...

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Abstract

The invention discloses a method for reducing the fluorescent background of a Taqman probe. The method comprises the following steps: when a solid-phase phosphoramidite triester method is adopted to synthesize the Taqman probe on a full-automatic DNA synthesizer, firstly capping a 3-terminal quenching group CPG, then carrying out a four-step circular reaction of deprotection, coupling, capping and oxidation, then treating a synthetic plate with a DEA-ACN mixed solution and ACN in sequence, then carrying out ammonolysis, and after the ammonolysis is finished, eluting a sample in the synthetic plate till a brand new 96-hole plate, and obtaining the probe through HPLC purification and separation. According to the method, the step of capping is added before the first-time deprotection in the four-step circular reaction of deprotection, coupling, capping and oxidation of a traditional solid-phase phosphoramidite triester method so as to prevent CPG which is not marked with quenching groups from participating in the subsequent reaction, and the existence of by-products with fluorescence labels is avoided, so that the fluorescence background of the Taqman probe is reduced.

Description

technical field [0001] The invention relates to the technical field of DNA synthesis, in particular to a method for reducing the fluorescence background of Taqman probes. Background technique [0002] As the core raw material of nucleic acid detection kits, Taqman probes require high quality to achieve detection accuracy, and have higher requirements for low background fluorescence and high sensitivity. The Taqman probe synthesis method in the present invention can better reduce the fluorescent background of the probe, improve sensitivity, and provide guarantee for efficient RT-PCR nucleic acid detection. [0003] At present, it is difficult for chemical products to be 100% pure. Therefore, there will be a small amount of unlabeled CPG in the 3-terminal quenching group CPG raw material of Taqman probe. Protection, coupling, capping, and oxidation are four-step cycle reactions, and the obtained Taqman probes will contain traces of fluorescently labeled by-products that are n...

Claims

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Application Information

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IPC IPC(8): C07H21/02C07H1/00C12Q1/6806
CPCC07H21/02C07H1/00C12Q1/6806C12Q2561/101Y02P20/55
Inventor 雍金贵刘宗文刘倩
Owner 通用生物(安徽)股份有限公司
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