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A high-fidelity polymerase with gap DNA preference and its application

A high-fidelity polymerase and preference technology, applied in the field of genetic engineering, can solve the problems of no substrate preference and lack of application value of polymerase, and achieve the effect of avoiding degradation

Active Publication Date: 2022-03-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In 2007, Heinz et al expressed and purified Deinococcus radiodurans in vitro ( Deinococcus radiodurans The protein (KlenDr) homologous to the E. coli Klenow fragment in R1) has reported its high-fidelity polymerization properties and weak strand replacement synthesis ability, but there is no further research on substrate preference, so the polymerase lacks Value

Method used

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  • A high-fidelity polymerase with gap DNA preference and its application
  • A high-fidelity polymerase with gap DNA preference and its application
  • A high-fidelity polymerase with gap DNA preference and its application

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Embodiment 1

[0025] Embodiment 1: Construction of Deinococcus radioduran KlenDr protein expression strain

[0026] According to the annotated Deinococcus radiodurans R1 genome (ASM163882v1) resequenced in 2016, DNA polymerase I (Protein Sequence ID: ANC71194.1) contains 921 amino acids, which is 35 amino acids less at the N-terminus than the old version (ASM856v1). The KlenDr involved in the present invention is derived from amino acids 289-921 of the new DNA polymerase I sequence. figure 1 is a schematic diagram of the domains of the DNA polymerase I family, in which the domain of Deinococcus radiodurans is located in the second item.

[0027] (1) The genome of Deinococcus radiodurans R1 was extracted using the Bacterial Genomic DNA Extraction Kit (DP302-02) of Tiangen Biotechnology, and its concentration and purity were determined with NanoDrop 1000 (Thermo Company).

[0028] (2) Design a pair of primers that can be used for homologous recombination according to amino acids 289-921 of t...

Embodiment 2

[0032] Embodiment 2: Induced expression of Deinococcus radiodurans KlenDr protein

[0033] (1) Transform the successfully constructed Pet28a-KlenDr expression vector into Escherichia coli BL21(DE3) expression strain (Quan Shi Jin Company), and use solid LB medium containing 40 μg / mL Kanamycin and 34 μg / mL chloramphenicol antibiotics to screen for successful transformation cloning strains.

[0034] (2) Pick a successfully transformed single colony into 5ml liquid medium, shake and culture overnight at 37°C, then transfer to 500ml medium, culture until OD600 is 0.6-0.8, put it on ice for 10min, add 100ul 1M IPTG was cultured at 30°C for 5h to induce the expression of the target protein.

[0035] (3) After induction, collect the cells by centrifugation at 8000rpm for 8 minutes, resuspend and wash the cells with 1x PBS, and finally centrifuge at 8000rpm for 5 minutes, discard the supernatant, and store the cells at -80°C.

Embodiment 3

[0036] Embodiment 3: the purification of Deinococcus radiodurans KlenDr protein

[0037] (1) Cell lysis: Add 15 mL of lysis buffer (300 mM NaCl, 20 mM Tris HCl pH 8.0, 5% Glycerol, 3 mM β-Me, 10 mM imidazole) to resuspend cells according to 1 g of cells (wet weight). Use an ultrasonic cell breaker (Ningbo Xinzhi, JY92-IIN) to break the cells in an ice-water bath until the suspension is transparent. The parameters are alternating rod Φ6, power 60%, ultrasound 2.5s, gap 9.9 s, time 60-90 min . The broken cell suspension was centrifuged at 15,000 rpm for 35 minutes to remove cell debris, and the supernatant was retained and filtered with a 0.22 or 0.45 μM filter membrane.

[0038](2) Nickel column affinity purification: Nickel column (HisTrap HP 1ml) was purchased from GE HealthCare. First use Ni-bufferA (300 mM NaCl, 20 mM Tris HCl pH 8.0, 5% Glycerol, 3 mM β-Me) to equilibrate the nickel column, use a flow rate of 1ml / min to fully combine the cell lysate with the nickel colum...

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Abstract

The invention discloses a gap DNA-preferential high-fidelity polymerase and its application. The invention discloses that the high-fidelity polymerization characteristic of the Klenow fragment (KlenDr) derived from the DNA polymerase I of Deinococcus radiodurans is not dependent on the 3'-5' corrective excision activity, and has a preference for binding to nick DNA, different Compatible with existing commercial high-fidelity polymerases. Due to the specific affinity of KlenDr to the gap DNA substrate, the 3' end of the upstream primer will not be excised, and the downstream nucleotide chain will rarely be replaced. It will have a very broad application prospect in genetic engineering operations such as gap filling and the construction of sequencing libraries.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a gap DNA-preferential high-fidelity polymerase and its application. Background technique [0002] DNA polymerase has a wide range of applications in genetic engineering operations such as molecular cloning, DNA sequencing, and library construction, and its ability to accurately replicate DNA sequences is crucial. Most of the high-fidelity DNA polymerases currently commercially used rely on the 3'-5' exo-correction activity: when the inserted nucleotide does not conform to the Watson-Crick base pairing principle, the inappropriate spatial conformation will force a new insertion The nucleotides are transferred from the polymerization active center to the 3'-5' excision active center, thereby excising the wrongly inserted nucleotides to ensure the accuracy of DNA replication. At present, there is a lack of high-fidelity polymerases that are not dependent on 3'-5' corrective excisio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12P19/34C12Q1/6806C40B50/06
CPCC12N9/1252C12Y207/07007C12P19/34C12Q1/6806C40B50/06C12Q1/6844C12Q2521/101C07K14/195
Inventor 华跃进周星茹王梁燕陈宣亦
Owner ZHEJIANG UNIV
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