PCR Cloning Method

  • My NEB
  • Print
  • PDF
  • Return to Cloning & Synthetic Biology

    PCR cloning differs from traditional cloning in that the DNA fragment of interest, and even the vector, can be amplified by the Polymerase Chain Reaction (PCR) and ligated together, without the use of restriction enzymes. PCR cloning is a rapid method for cloning genes, and is often used for projects that require higher throughput than traditional cloning methods can accommodate. It allows for the cloning of DNA fragments that are not available in large amounts.

    Typically, a PCR reaction is performed to amplify the sequence of interest, and then it is joined to the vector via a blunt or single-base overhang ligation prior to transformation. Early PCR cloning often used Taq DNA Polymerase to amplify the gene. This results in a PCR product with a single template-independent base addition of an adenine (A) residue to the 3' end of the PCR product, through the normal action of the polymerase. These "A-tailed" products are then ligated to a complementary T-tailed vector using T4 DNA ligase, followed by transformation. 

    1. How Does the NEB PCR Cloning Kit Work?

      What are toxic mini-genes, and how do they improve transformation efficiencies? Becky explains.

    2. Behind the Product: The NEB® PCR Cloning Kit

      For the inside scoop on how NEB products come to be, learn the story behind the new NEB® PCR Cloning Kit.

    3. Overview of PCR Cloning

      PCR Cloning is an easy and reliable cloning method. The name is derived from the use of a DNA amplification step to generate the amplicon. Learn more about the benefits and disadvantages of PCR Cloning.

    High-fidelity DNA polymerases are also now routinely used to amplify sequences with the PCR product containing no 3' extensions. The blunt-end fragments are joined to a plasmid vector through a typical ligation reaction or by the action of an "activated" vector that contains a covalently attached enzyme, typically Topoisomerse I, which facilitates the vector:insert joining. Some PCR cloning systems contain engineered "suicide" vectors that include a toxic gene into which the PCR product must be successfully ligated to allow propagation of the strain that takes up the recombinant molecule during transformation.

    A typical drawback common to many PCR cloning methods is a dedicated vector that must be used. These vectors are typically sold by suppliers, like NEB, in a ready-to-use linearized format and can add significant expense to the total cost of cloning. Also, the use of specific vectors restricts the researcher's choice of antibiotic resistance, promoter identity, fusion partners, and other regulatory elements.

    PCR Cloning

    Note that times are based on estimates for moving a gene from one plasmid to another. If the source for gene transfer is gDNA, add 2 hours to calculation for the traditional cloning method. Total time does not include transformation, isolation or analysis.

    Featured Products:
    Monarch™ Plasmid Miniprep Kit
    Monarch™ DNA Gel Extraction Kit
    Monarch™ PCR & DNA Cleanup Kit (5 μg)
    NEB PCR Cloning Kit
    Q5® High-Fidelity DNA Polymerase
    NEB 5-alpha Competent E.coli
    Blunt/TA Ligase Master Mix

    Find Additional Products for use in PCR Cloning applications:
    DNA Preparation
    Restriction Enzyme Digestion
    Reverse Transcription (cDNA Synthesis)
    DNA End Modification
    Phoshporylation (Kinase)
    Cloning Ligation
    DNA Isolation
    DNA Analysis
    Restriction Enzyme Digestion
    Colony PCR
    DNA Sequencing