Home FAQs Which DNA polymerase is best for my isothermal amplification reaction?

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
  OPTIMAL TEMPERATURE 3' to 5' EXO ACTIVITY 5' to 3' EXO ACTIVITY AMPLIFICATION
SPEED
 ROOM
TEMPERATURE
SETUP
 REVERSE
TRANSCRIPTASE
ACTIVITY
 INHIBITOR
TOLERANCE
 APPLICATIONS

phi29
30
***
N/A
**
N/A
N/A
*		 Whole-genome amplification, multiple displacement amplification
Bsu DNA Polymerae, Large Fragment
37-45
N/A
N/A
**
N/A
*
*		 Isothermal amplification and strand displacement at low temperature (37-45C)
Bst DNA Polymerase,
Full Length
50-65
N/A
**
N/A
N/A
N/A
*		 Nick translation reactions at elevated temperatures
Bst DNA Polymerase,
Large Fragment

50-65
N/A
N/A
*
N/A
*
*		 General strand-displacement reactions, original polymerase for LAMP and other diagnostic amplifications
Bst 2.0
DNA Polymerase
50-65
N/A		  
N/A
**		  
N/A
**
**		 Improved LAMP, SDA, and other amplification reactions
Bst 2.0 WarmStart
DNA Polymerase
55-65
N/A		  
N/A
**
***
**
**		 Consistent, room-temperature, and high-throughput amplification assays
Bst 3.0
DNA Polymerase
55-72
N/A		  
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