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Related Concept Videos

Nucleotide Excision Repair01:08

Nucleotide Excision Repair

Overview
Nucleotide Excision Repair01:38

Nucleotide Excision Repair

DNA Distortion and Damage
Cells are regularly exposed to mutagens—factors in the environment that can damage DNA and generate mutations. UV radiation is one of the most common mutagens and is estimated to introduce a significant number of changes in DNA. These include bends or kinks in the structure, which can block DNA replication or transcription. If these errors are not fixed, the damage can cause mutations, which in turn can result in cancer or disease depending on which sequences are...
DNA Isolation01:24

DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
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.
The first step of...
Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

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...
Homologous Recombination02:31

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...

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CRISPR-based Shuttle Cloning: A High-throughput Cloning Method
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DNA cloning and engineering by uracil excision.

Jurate Bitinaite1, Nicole M Nichols

  • 1New England Biolabs, Ipswich, Massachusetts, USA.

Current Protocols in Molecular Biology
|April 4, 2009
PubMed
Summary
This summary is machine-generated.

This study presents a simple DNA engineering method using deoxyuracil (dU) primers for seamless gene assembly and modification. This approach enables efficient, single-format experiments for creating customized DNA molecules and introducing mutations.

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Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Biotechnology

Background:

  • Traditional DNA engineering methods can be complex and time-consuming.
  • Efficient assembly of multiple DNA fragments and introduction of mutations are crucial for genetic studies.

Purpose of the Study:

  • To describe a simple and efficient DNA engineering method.
  • To enable seamless, directional assembly of multiple DNA fragments and nucleotide sequence alteration in a single experiment.

Main Methods:

  • Utilizing PCR primers containing a single deoxyuracil (dU) residue.
  • Amplifying DNA fragments incorporating dU at each end.
  • Employing uracil excision to generate overlapping single-stranded extensions for seamless assembly.

Main Results:

  • Developed a method for nucleotide sequence alteration, multiple PCR fragment assembly, and directional cloning.
  • Demonstrated the ability to create customized DNA molecules and perform multi-fragment assemblies in a single-format experiment.
  • Provided protocols for primer design, uracil excision-based engineering, and vector preparation.

Conclusions:

  • The deoxyuracil-based DNA engineering method offers a simple and efficient approach for constructing customized DNA molecules.
  • This technique facilitates both complex DNA assembly and precise mutagenesis, streamlining genetic engineering workflows.