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Optimized polymerase for amplifying target nucleic acid, composite system and application

A technology of polymerase and system, applied in the field of optimized amplification and purification of target nucleic acid

Active Publication Date: 2020-07-28
APOGENOMICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Regarding how to find a suitable substrate to amplify DNA of indefinite length by primer extension method under the premise of retaining the 3'-5'exonuclease activity of DNA polymerase, especially for the application scenario of single-strand linear amplification technology, now Some technologies do not provide technical inspiration

Method used

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  • Optimized polymerase for amplifying target nucleic acid, composite system and application
  • Optimized polymerase for amplifying target nucleic acid, composite system and application
  • Optimized polymerase for amplifying target nucleic acid, composite system and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1. Matrix experiments of different DNA polymerases and different biotin-dNTP analogs

[0104] Purpose

[0105] Through the cross-use matrix experiment of different modified biotin-dNTPs and different DNA polymerases under the same mixing ratio, the amplification performance under the combination of different DNA polymerases and different dNTP analogs was evaluated, and the enzymes and enzymes that met the amplification and purification requirements were screened. dNTP analogs.

[0106] The DNA polymerase protein sequence used in this example is shown in Table 1.

[0107] Table 1. DNA polymerase protein sequences

[0108]

[0109]

[0110]

[0111] The oligonucleotide sequences used in this example are shown in Table 2.

[0112] Table 2. Oligonucleotide sequences

[0113]

[0114] Wherein the 3'-OH of Primer 2 (SEQ ID NO: 11) is replaced with C3 Spacer.

[0115] Main reagents and materials

[0116] The free peripheral blood DNA extraction kit us...

Embodiment 2

[0156] Example 2. Optimization of the Mixing Ratio of Biotin-dNTP Analogs and Mixing and Incorporation Experiments of Various Biotin-dNTP Analogs

[0157] Purpose

[0158] Several biotin-dNTP analogs with better incorporation effects screened out in Example 1 were optimized for mixing ratio and incorporation. According to the performance of selected biotin-dNTP analogs under the catalysis of selected DNA polymerases under different incorporation gradients and mixed incorporation conditions, the mixing ratio and mixed incorporation conditions of biotin-dNTP analogs were optimized.

[0159] Experimental materials and methods

[0160] The experimental materials and equipment used in this example are exactly the same as those in Example 1, the only difference being the mixing ratio of the dNTP mixture.

[0161] Using biotin-14-dATP, biotin-11-dGTP, and biotin-14-dCTP with an initial concentration of 1 mM, they were prepared to account for 10%, 20%, 50%, and 80% of dATP, dGTP, an...

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Abstract

The invention relates to the field of a biological technology, and discloses an optimized method for amplifying target nucleic acid. The method comprises the steps of enabling a formwork combined withprimers, at least part of dNTP modified with tagged molecules, and high fidelity polymerase DNA polymerase having 3'-5' excision enzyme activity to be in contact to obtain amplification products, soas to obtain purified products of which the yield and the tagged molecule incorporation efficiency are ideal. The invention further discloses a dNTP-DNA polymerase composite system having high incorporation efficiency for the method, DNA polymerase which is reformed, and the dNTP modified with the tagged molecules. According to the method and the composite system provided by the invention, the reformed DNA polymerase, the dNTP modified with the tagged molecules, and single strands of different lengths are subjected to amplification, so that notable technological progress is realized, and the method has broad application prospects and notable economic value.

Description

technical field [0001] The present invention relates to the field of biotechnology, more specifically, relates to an optimized method and system for amplifying and purifying target nucleic acid. Background technique [0002] For targeted amplification technology, it is critical to obtain target products with sufficient yield and purity after amplification and purification. In the prior art, the purification methods for target products after nucleic acid amplification mainly include solid-phase carrier adsorption methods, such as adsorption column method or solid-phase reversible magnetic bead method, which utilize the strong affinity and adsorption force of the carrier to nucleic acid itself; molecular sieve method , to screen target molecules through the difference in molecular weight; electrophoresis, through electrophoretic analysis, to purify and recover nucleic acids with target molecular weights; affinity purification, to label probes or primers with markers, and use s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C07H21/04C12Q1/686
CPCC12N9/1252C07H21/04C12Q1/686C12Y207/07007C12Q2521/101C12Q2525/10
Inventor 杨国华郭志伟林国旻车彬余佳佳李杰
Owner APOGENOMICS CO LTD