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