Thermostable Taq enzyme temperature control affinity ligand, and preparation method and application thereof

A thermostable and affinity technology, applied in biochemical equipment and methods, transferases, viruses/phages, etc., can solve the problems of noise, imperfect fidelity, hybrid bands, etc., to avoid primer-dimers, hybrids, etc. Low-band, low-noise effect

Active Publication Date: 2018-01-23
INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, the technical problem to be solved in the present invention is to overcome the problems of banding, noise and imperfect fidelity in the DNA polymerase chain reaction of existing hot-start high-temperature-resistant DNA polymerases, thereby providing a thermostable Taq enzyme Temperature-controlled affinity ligand and its preparation method and application

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  • Thermostable Taq enzyme temperature control affinity ligand, and preparation method and application thereof
  • Thermostable Taq enzyme temperature control affinity ligand, and preparation method and application thereof
  • Thermostable Taq enzyme temperature control affinity ligand, and preparation method and application thereof

Examples

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Embodiment 1

[0046] Example 1: Preparation of thermostable Taq enzyme temperature-controlled affinity ligand

[0047] A method for preparing a thermostable Taq enzyme affinity ligand described in this embodiment comprises the following steps:

[0048] 1. Coating and sealing of ELISA plate: Dilute the concentration of Taq enzyme protein to 100 μg / mL with coating buffer, and use it as the coating solution for the first round of panning, and for the second to fourth rounds of panning The concentrations of the coating solution diluted according to the above method are: 50 μg / mL, 25 μg / mL and 10 μg / mL. After the ELISA plate was irradiated by 15W UV lamps at a distance of 10cm for 15mim, the coating solution prepared above was divided into four wells at 100μL / well, and two pretreatment wells were coated with pretreatment well buffer, and placed in a self-sealing Incubate in the bag overnight at 4-8°C, discard the coating solution, wash the plate 4 times with TBST, each time for 3 minutes, pat d...

Embodiment 2

[0058] Example 2: Determination of the Amino Acid Sequence of Taq Enzyme Temperature Control Affinity Ligand

[0059] The phages panned in Example 1 were cloned and amplified, the single-stranded DNA of the phages was dissolved and extracted with sodium iodide, identified by agarose gel electrophoresis and PCR, and then sequenced. According to the reading frame of the phage pIII gene in the coding chain, the amino acid sequence of the foreign polypeptide fused with the pIII protein is deduced to be H-Thr-Trp-Leu-His-Phe-Pro-His-OH, which is the amino acid sequence of the ligand As shown in SEQ ID NO:1.

Embodiment 3

[0060] Embodiment 3: the preparation of whole process hot start Taq enzyme

[0061] Mix the 1000-fold diluted filtrate in Step 10 of Example 1 with 10 μg / μL Taq enzyme in equal volumes to form a full-range hot-start Taq enzyme.

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Abstract

The invention belongs to the technical field of whole-course heat start Taq enzyme preparation, and discloses thermostable Taq enzyme temperature control affinity ligand, and preparation method and application thereof. The amino acid sequence of the ligand is as shown in SEQ ID NO:1, the ligand is thermostable under the condition that the temperature is less than or equal to 96 DEG C, the closed or opened Taq enzyme activity is reversible when the temperature is less than or equal to 96 DEG C, the ligand can take part in heat start of the Taq enzyme in the whole course in the whole PCR circulation, Taq enzyme heat start is conducted only when a primer and a template are scrupulously matched in each circulation process, non-specific amplification and generation of a primer dimer are avoided, reaction specificity and sensitivity are improved, the DNA polymerase chain reaction result has few impurities and low noise, and the fidelity is perfect; meanwhile, the ligand adopts a short peptide structure, has smaller molecular weight and smaller PCR interference compared with those of an antibody, is not like single chain DNA which is easy to degrade, and the whole-course heat start Taq enzyme product prepared by the ligand has higher stability.

Description

technical field [0001] The invention belongs to the technical field of hot-start Taq enzyme preparation, and in particular relates to a heat-stable Taq enzyme temperature-controlling affinity ligand and a preparation method and application thereof. Background technique [0002] Hot-start Taq enzyme, also known as hot-start high-temperature-resistant DNA polymerase, has been increasingly used in PCR technology, especially in multi-site gene multiplex amplification technology, such as in microsatellite genes of animals and plants, human Individual identification or paternity analysis. Compared with ordinary Taq enzymes, the biggest feature of this enzyme is that the enzymatic activity of the hot-start Taq enzyme is released and restored during the reaction system from high temperature (95°C) to the annealing temperature, that is, in the reaction system, the primer and The template is in a stringent paired binding state to start PCR amplification, so this amplification is a mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C12N9/12C12N7/01
Inventor 涂祖新郑国华熊勇华陈媛张莉莉刘卓荣杜建华
Owner INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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