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Related Experiment Videos

DNA cloning using in vitro site-specific recombination.

J L Hartley1, G F Temple, M A Brasch

  • 1Life Technologies, Inc., Rockville, Maryland 20850, USA. jhartley@lifetech.com

Genome Research
|November 15, 2000
PubMed
Summary

Recombinational cloning enables efficient, high-throughput gene functional analysis by rapidly subcloning DNA segments into various vectors. This method facilitates protein expression optimization and the creation of translation fusions.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Genome sequencing projects identify numerous genes with unknown functions.
  • Analyzing these genes requires efficient DNA manipulation for protein expression and functional studies.

Purpose of the Study:

  • To introduce and describe recombinational cloning, a novel method for directional gene cloning.
  • To demonstrate its utility in high-throughput functional analysis and protein expression optimization.

Main Methods:

  • Utilizes in vitro site-specific recombination for directional cloning of PCR products.
  • Enables automatic, high-efficiency subcloning of DNA segments into diverse vector backbones.

Main Results:

  • Recombinational cloning allows parallel transfer of numerous DNA segments into multiple vectors.

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  • Subclones maintain DNA orientation and reading frame register, enabling fusion protein generation.

    • Conclusions:

    • Recombinational cloning offers a powerful approach for high-throughput gene analysis.
    • It accelerates the optimization of protein expression and facilitates the study of gene function.