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Related Concept Videos

Maxam-Gilbert Sequencing01:05

Maxam-Gilbert Sequencing

In the same year as the discovery of the Sanger sequencing method, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their chemical-cleavage method for DNA sequencing. The Maxam-Gilbert method relies on using different chemicals that can cleave the DNA sequence at specific sites, the separation of resulting DNA fragments of variable size using electrophoresis, and deciphering the DNA sequence from the resulting gel bands.
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Sanger Sequencing

DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
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Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
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Base Excision Repair01:54

Base Excision Repair

One of the common DNA damages is the chemical alteration of single bases by alkylation, oxidation, or deamination. The altered bases cause mispairing and strand breakage during replication. This type of damage causes minimal change to the DNA double helix structure and can be repaired by the base excision repair (BER) pathways. BER corrects damaged DNA sequences by removing the damaged base and restoring the original base sequence using the complementary strand as a template.
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Restriction Enzymes01:11

Restriction Enzymes

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.
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Homologous Recombination

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Related Experiment Video

Updated: May 13, 2026

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly
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Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly

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DNA fragments assembly based on nicking enzyme system.

Rui-Yan Wang1, Zhen-Yu Shi, Ying-Ying Guo

  • 1MOE Key Lab of Bioinformatics and Systems Biology, Department of Biological Science and Biotechnology, School of Life Sciences, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China.

Plos One
|March 14, 2013
PubMed
Summary

We developed Nicking Endonuclease-based Ligation-Independent Cloning (NE-LIC) for efficient DNA assembly. This method generates single-stranded DNA overlaps, optimizing fragment assembly for synthetic biology applications.

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Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Metabolic Engineering

Background:

  • DNA ligation-independent cloning (LIC) is crucial for assembling DNA fragments.
  • Existing LIC methods have limitations in certain applications.

Purpose of the Study:

  • To introduce a novel NEase-based LIC (NE-LIC) method.
  • To optimize NE-LIC for efficient DNA fragment assembly.
  • To demonstrate NE-LIC's utility in metabolic engineering.

Main Methods:

  • Generating single-stranded DNA (ssDNA) overlaps using Nicking Endonucleases (NEases).
  • Optimizing factors influencing cloning efficiency, including overlap length.
  • Comparing NE-LIC with existing methods like SLIC and USER.

Main Results:

  • NE-LIC effectively generates 3' or 5' ssDNA overlaps of various lengths.
  • Optimal assembly efficiency was observed with 10 bp/15 bp overlaps, and 5 bp/10 bp overlaps with T4 DNA ligase.
  • NE-LIC demonstrated clear advantages over SLIC and USER.
  • Successfully assembled a six-gene pathway for poly-3-hydroxybutyrate (PHB) synthesis.

Conclusions:

  • NE-LIC provides a versatile and efficient approach for DNA assembly.
  • The NEase-based mechanism enhances LIC strategies.
  • NE-LIC is applicable to complex metabolic engineering tasks, such as PHB production.