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Related Concept Videos

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.
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
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...
Recombinant DNA01:09

Recombinant DNA

Overview
RACE - Rapid Amplification of cDNA Ends02:35

RACE - Rapid Amplification of cDNA Ends

Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
Since the...

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Related Experiment Video

Updated: May 8, 2026

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
09:02

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Published on: January 8, 2015

Restrictionless cloning.

Mikkel A Algire1

  • 1J. Craig Venter Institute, Synthetic Biology Group, Rockville, MD, USA.

Methods in Enzymology
|September 10, 2013
PubMed
Summary

Restrictionless cloning offers efficient, directional cloning of PCR products into vectors. This method bypasses the need for specific restriction sites on inserts, simplifying molecular cloning workflows.

Area of Science:

  • Molecular Biology
  • Gene Cloning
  • Recombinant DNA Technology

Background:

  • Traditional PCR product cloning methods like blunt-end, TA cloning, and restriction site-based cloning have limitations.
  • These methods often require specific vector sites or primer modifications, adding complexity to molecular cloning.
  • Efficient and versatile cloning strategies are crucial for various molecular biology applications.

Purpose of the Study:

  • To introduce and evaluate a novel restrictionless cloning technique.
  • To demonstrate the efficiency and versatility of restrictionless cloning for directional PCR product insertion.
  • To provide a simplified alternative to existing PCR cloning methods.

Main Methods:

  • Developed a restrictionless cloning strategy for Polymerase Chain Reaction (PCR) products.
Keywords:
Denaturation and annealingLigation and transformationPolymerase chain reaction (PCR)Primer designRestrictionless cloningVector preparation

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  • Applied the technique to directionally clone PCR products into various vector sites.
  • Assessed the efficiency of the cloning method without relying on insert- or vector-specific restriction sites.

    • Main Results:

    • The restrictionless cloning technique demonstrated high efficiency in directional cloning of PCR products.
    • The method successfully cloned PCR products into any available cloning site within a vector.
    • Cloning efficiency was maintained regardless of the presence or absence of restriction sites within the PCR insert.

    Conclusions:

    • Restrictionless cloning provides an efficient and versatile method for directional cloning of PCR products.
    • This technique simplifies the molecular cloning process by eliminating the requirement for specific restriction sites.
    • The developed method offers a valuable alternative for researchers seeking streamlined gene cloning strategies.