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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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Updated: Feb 18, 2026

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
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DNA複製フォークで精密な編集により,ユカリオットの複合ゲノム工学が可能になる

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
まとめ

この研究は,酵母における新しいマルチプレックスゲノム工学の方法を紹介しています. 精密で効率的なDNA改変を 2本鎖の断絶なしに可能にし 経路工学のための広範な遺伝的多様性を生み出します

キーワード:
DNA複製ラッド51ゲノム編集同型再結合メタボリックエンジニアリングマルチプレックスゲノム工学自然産物ssDNA オリゴデオキシヌクレオチド

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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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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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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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Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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科学分野:

  • 分子生物学
  • 合成生物学
  • 遺伝学

背景:

  • 現在のゲノム工学の方法は,しばしば二重鎖の断裂と同種の再結合に依存しており,これは意図しない変異につながる可能性があります.
  • マルチプレックスゲノム工学は 複雑な遺伝的多様性を効率的に生み出すのに不可欠です

研究 の 目的:

  • Saccharomyces cerevisiaeにおける新しい,効率的で正確なマルチプレックスゲノムエンジニアリング技術を開発する.
  • 生物合成経路の組み合わせによる多様化のためのこの技術の能力を実証する.

主な方法:

  • 合成オリゴヌクレオチドをDNA複製の遅延糸で利用した.
  • Rad51指向の同型再結合と二重鎖DNA破裂の必要性を回避した.
  • 複数のオリゴヌクレオチドと標的型変異の同時組み込みを達成した.

主要な成果:

  • >40%の効率で単一の塩基対解像度で精密な染色体改変が実証されています.
  • 一つの変換で最大12のオリゴヌクレオチドと60の変異を成功裏に組み込みました
  • 繰り返し変換によって生成された 10^5を超える組み合わせゲノム多様性.
  • 精密な変異により変異したカロテノイドレベルを持つ変種を生成する異質のβ-カロテンの生物合成経路を設計した.

結論:

  • 開発された方法は,マルチプレックスゲノム工学のためのRad51独立の,二重鎖のブレイクフリーアプローチを提供します.
  • この技術は高効率で精密で組み合わせられた ユーカリオットゲノムの改変を可能にします
  • この戦略は自動化可能で,代謝工学を含む様々な用途に重要なゲノム多様性を生み出すために適用できます.