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Related Experiment Videos

Complete open reading frame (C-ORF) technology: simple and efficient technique for cloning full-length protein-coding

Dong-Chul Kang1, Paul B Fisher

  • 1Ilsong Institute of Life Science, Hallym University, 1605-4, Kwanyang-dong, Dongan-gu, Anyang, Kyonggi-do, Republic of Korea.

Gene
|June 2, 2005
PubMed
Summary

Researchers developed the C-ORF technique using a degenerate stem-loop annealing primer (dSLAP) for efficient full-length cDNA cloning. This method simplifies the process of identifying complete open reading frames from expressed sequence tags.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Full-length cDNA cloning is crucial for characterizing expressed sequence tags (ESTs).
  • Existing technical challenges in cDNA cloning limit the identification of novel genes.
  • Complete open reading frame (C-ORF) identification is essential for functional genomics.

Purpose of the Study:

  • To present improved methods for efficient full-length cDNA cloning.
  • To introduce the C-ORF technique utilizing a novel degenerate stem-loop annealing primer (dSLAP).
  • To enable the cloning of complete open reading frames from various cDNA targets.

Main Methods:

  • Development of the C-ORF technique based on the polymerase chain reaction (PCR).
  • Utilizing a degenerate stem-loop annealing primer (dSLAP) for specific cDNA annealing.

Related Experiment Videos

  • A three-step protocol: reverse transcription, dSLAP annealing with second-strand synthesis, and PCR amplification.

    • Main Results:

    • Successful cloning of complete open reading frames for both known and unknown cDNA targets.
    • High efficiency, with most targets cloned after a single application of the C-ORF method.
    • Demonstrated applicability to cDNA targets with limited or incomplete sequence information.

    Conclusions:

    • The C-ORF technique offers a simple and effective solution for full-length cDNA cloning.
    • The dSLAP primer design effectively suppresses internal second-strand synthesis.
    • This method is broadly applicable and accessible for individual laboratories, advancing gene characterization.