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相关概念视频

Replication in Eukaryotes02:31

Replication in Eukaryotes

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Overview
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Replication in Eukaryotes01:29

Replication in Eukaryotes

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In eukaryotic cells, DNA replication is highly conserved and tightly regulated. Multiple linear chromosomes must be duplicated with high fidelity before cell division, so there are many proteins that fulfill specialized roles in the replication process. Replication occurs in three phases: initiation, elongation, and termination, and ends with two complete sets of chromosomes in the nucleus.
Many Proteins Orchestrate Replication at the Origin
Eukaryotic replication follows many of the same...
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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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The DNA Replication Fork01:02

The DNA Replication Fork

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An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork.   Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication...
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The DNA Replication Fork01:02

The DNA Replication Fork

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Proofreading01:31

Proofreading

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Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
Errors During Replication are Corrected by the DNA Polymerase...
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相关实验视频

Updated: Feb 18, 2026

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
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Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

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在DNA复制叉上精确编辑使得真核生物中的多重基因组工程成为可能

Edward M Barbieri1, Paul Muir1, Benjamin O Akhuetie-Oni1

  • 1Department of Molecular, Cellular, & Developmental Biology, Yale University, New Haven, CT 06520, USA; Systems Biology Institute, Yale University, West Haven, CT 06516, USA.

Cell
|November 21, 2017
PubMed
概括

这项研究引入了一种新的多重基因组工程方法. 它可以在没有双链断裂的情况下进行精确,高效的DNA修改,为路径工程创造广泛的遗传多样性.

关键词:
DNA复制拉德51基因组编辑同类重组代谢工程多重基因组工程自然产品ssDNA寡氧核酸

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A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells
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Genome Editing in Mammalian Cell Lines using CRISPR-Cas
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相关实验视频

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A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells
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科学领域:

  • 分子生物学
  • 合成生物学
  • 遗传学

背景情况:

  • 目前的基因组工程方法通常依赖于双链断裂和同源重组,这可能导致意外突变.
  • 多重基因组工程对于有效地创造复杂的遗传多样性至关重要.

研究的目的:

  • 在Saccharomyces cerevisiae中开发一种新,高效和精确的多重基因组工程技术.
  • 展示该技术在生物合成途径的组合多样化方面的能力.

主要方法:

  • 在DNA复制的滞后链上利用合成寡核酸的化.
  • 绕过了对Rad51指导的同源重组和双链DNA断裂的需要.
  • 实现了多个寡核酸和向突变的同时结合.

主要成果:

  • 在单个基因对分辨率上证明了精确的染色体修饰,效率高于40%.
  • 在一次转变中成功结合了12个寡核酸和60个突变.
  • 通过代转换产生超过10^5的组合基因组多样性.
  • 设计了一种异质β-胡卜素生物合成途径,产生由于精确突变而改变的胡卜素水平的变体.

结论:

  • 开发的方法为多重基因组工程提供了Rad51独立的双链无断方法.
  • 这项技术使得真核生物基因组的高效,精确和组合性修改成为可能.
  • 该策略可自动化,可用于产生各种应用的显著基因组多样性,包括代谢工程.