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相关概念视频

Restriction Enzymes01:11

Restriction Enzymes

30.7K
Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. Such palindromic DNA sequences or inverted repeats are commonly found in regions of functional significance, such as the origin of replication, gene operator sites, and regions containing transcription termination signals.
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
30.7K
Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
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相关实验视频

Updated: Jul 1, 2025

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

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使用I-SceI的可编程内核酶活动的积极选择屏幕.

Michael A Mechikoff1, Kok Zhi Lee2, Kevin V Solomon3

  • 1Department of Biology, US Air Force Academy, Colorado Springs, CO, USA.

Methods in molecular biology (Clifton, N.J.)
|March 12, 2024
PubMed
概括

我们开发了一个基于I-SceI的新平台,用于灵活选DNA内核酶. 这种方法丰富了更强大的酶变体,克服了严格的选择方法的局限性.

关键词:
克里斯普尔是什么意思?克里斯普尔是什么意思?定向进化是指导进化的.丰富方式 丰富方式我-选择了积极的选择选择是积极的选择.细胞内核酶 (endonuclease) 是一种细胞内核酶.

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Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells
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Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

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Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
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相关实验视频

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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

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Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells
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Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
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科学领域:

  • 生物化学 生物化学
  • 分子生物学分子生物学
  • 酵素工程是什么? 酶工程是什么

背景情况:

  • 高通量选对于酶的发现和工程至关重要.
  • 现有的阳性选择幕可能过于严格,导致高假负率.
  • 新型可编程DNA内核酶需要灵活且不那么严格的选平台.

研究的目的:

  • 开发一种基于I-SceI的新平台,用于选可编程DNA内核酶.
  • 与现有方法相比,创建一个更灵活,更不严格的选择系统.
  • 为了能够有效地表征和丰富活跃的内核酶变体.

主要方法:

  • 在大肠杆菌中开发了一个基于I-SceI的系统,其中基因组分裂抑制了生长.
  • 通过塑固化或I-SceI表达等离子体被候选内核酶分裂来利用活动依赖的生长救援.
  • 用Cas9展示了该平台,并将其适应各种可编程DNA内核酶.

主要成果:

  • 该平台允许对内核酶候选物的活性依赖的丰富.
  • 更活跃的变种优先繁殖,导致成功的丰富.
  • 该系统有效地表征单个候选人,并丰富了图书馆的变体.

结论:

  • 这种基于I-SceI的新平台提供了一种灵活而强大的工具,用于选DNA内核酶.
  • 这种系统克服了严格选择的局限性,减少了假阴性.
  • 该协议可以适应各种内核酶和生物多样性开采和定向进化等应用.