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  • FAQ: Which DNA polymerase is best for my isothermal amplification reaction?

    Strand-displacement activity is essential to most isothermal techniques, and all of our relevant DNA polymerases exhibit strong strand-displacement activity. The most important parameter for choosing an isothermal polymerase is desired reaction temperature.

    Many isothermal applications use elevated temperature (> 50°C) and, for these, a thermophilic DNA polymerase is required. The large fragment of Bacillus stearothermophilus polymerase I (Bst DNA Polymerase, Large Fragment; NEB #M0275) has long been the enzyme of choice for these applications, however, NEB has recently developed an improved version of this polymerase, Bst 2.0 (NEB #M0537) for more robust isothermal amplification. For high-throughput and diagnostic applications requiring room-temperature setup and high reproducibility we recommend the WarmStart® version of Bst 2.0 (NEB #M0538). Further improvements in amplification speed, inhibitor tolerance, and reverse transcriptase produce our latest version, Bst 3.0 (NEB #M0374), recommended for the most robust isothermal and single-enzyme RT-LAMP reactions. If 5′ to 3′ exonuclease activity, rather than strand displacement activity, is desired, we also offer the full-length Bst DNA Polymerase (NEB #M0328) for these applications.

    Our mesophilic DNA polymerases are appropriate for lower temperature reactions. The exo- variant of the E. coli DNA Polymerase I Klenow fragment (NEB #M0212) and large fragment of Bsu DNA Polymerase (NEB #M0330) are good options for many of these applications. If extreme processivity or proofreading exonuclease activity are desired, phi29 DNA Polymerase (NEB #M0269) is the enzyme of choice.

    NEB's Selection of Bst DNA Polymerase-based Products for Isothermal DNA Amplification
      5′ to 3′
    EXO ACTIVITY
    AMPLIFICATION
    SPEED
    ROOM
    TEMPERATURE
    SETUP
    REVERSE
    TRANSCRIPTASE
    ACTIVITY
    INHIBITOR
    TOLERANCE
    APPLICATIONS
    Bst DNA Polymerase,
    Full Length

    **

    N/A

    N/A

    N/A

    *
    Nick translation reactions at elevated temperatures
    Bst DNA Polymerase,
    Large Fragment

    N/A

    *

    N/A

    *

    *
    General strand-displacement reactions, original polymerase for LAMP and other diagnostic amplifications
    Bst 2.0
    DNA Polymerase
     
    N/A

    **
     
    N/A

    **

    **
    Improved LAMP, SDA, and other amplification reactions
    Bst 2.0 WarmStart
    DNA Polymerase
     
    N/A

    **

    ***

    **

    **
    Consistent, room-temperature, and high-throughput amplification assays
    Bst 3.0
    DNA Polymerase
     
    N/A

    ***

    **

    ***

    ***
    Fastest, most robust LAMP and RT-LAMP reactions. High reverse transcriptase activity up to 72°C

    ***   Optimal, recommended product for selected application
     **   Works well for selected application
      *    Will perform selected application, but is not recommended
    N/A  Not applicable to this application