Loop-mediated Isothermal Amplification (LAMP)
Overview:
LAMP is an isothermal amplification method designed to detect a target nucleic acid without requiring sophisticated equipment. LAMP provides high sensitivity (to fg or <10 copies of target) but with rapid results: reactions can be performed in as little as 5–10 minutes. Reactions can be performed with limited resources, using a water bath for incubation and detection of results by eye, or with real-time measurement and high-throughput instruments. Detection of RNA targets is accomplished by simple addition of a reverse transcriptase to the LAMP reaction, with RT-LAMP performed as a true one-step, isothermal workflow.Required Materials:
- Bst 3.0 (M0374), Bst 2.0® (M0537), Bst 2.0 WarmStart® (M0538), or wild-type Bst DNA polymerase, large fragment (M0275)
- WarmStart® RTx Reverse Transcriptase (M0380)
- 10X Isothermal Amplification Buffer (Bst 2.0) or Thermopol Buffer (Bst)
- 10 mM dNTP Mix (N0447)
- 100 mM MgSO4 (B1003S)
- LAMP primers (note: we strongly recommend using NEB LAMP Primer Design Tool)
- Heatblock or waterbath, set to 65 °C
Optional Materials:
- Real-time fluorimeter (qPCR machine) or turibidimeter for real-time monitoring
- SYTO®-9 for real-time fluorescence
- Calcein, hydroxynaphthol blue, malachite green, or SYBR® Green 1 for endpoint analysis
- dUTP and Thermolabile Uracil DNA Glycosylase (M0372 ) for carryover contamination prevention (note: Bst 2.0 is strongly recommended for use with dUTP/UDG systems)
Before You Start:
A LAMP Primer mix can be prepared with all 4 or 6 (with Loop) primers. A 10X Primer Mix should contain: 16 µM FIP, 16 µM BIP, 2 µM F3, 2 µM BE, 4 µM LoopF, 4 µM LoopB in TE or water.Reactions should be setup on ice. If room temperature setup is desired, use Bst 2.0 WarmStart DNA polymerase.
If analyzing via agarose gel electrophoresis or other method requiring opening LAMP reaction vessels, setup secondary analysis area and equipment to avoid contamination.
Procedure:
| Component | Volume (25 µl reaction) |
| 10X Isothermal Amplification Buffer | 2.5 µL |
| 10 mM dNTPs | 3.5 µL (1.4 mM final) |
| 100 mM MgSO4 | 1.5 µL (6 mM+2 mM in buffer=8 mM final) |
| 10X Primers | 2.5 µL (1.6 µM FIP/BIP, 0.2 µM F3/B3, 0.4 µM LoopF/B) |
| Bst 2.0 (8,000 U/mL) | 1 µL (0.32 U/µL) |
| Sample | e.g. 1µL |
| H2O | 13 µL |
| For RNA Targets: WS RTx | 0.5 uL |
Running a no-template control is strongly recommended to ensure amplification specificity.
If optimization is desired, try titrating concentration of Mg (4–10 mM final) or Bst 2.0 (0.04-0.32 U/µL), or changing reaction temperature (50–72 °C).
References:
Notomi et al. (2000) Nucleic Acids Res 28(12): E63.Hsieh et al. (2014) Chem Commun 50(28): 3747-9.
Tanner and Evans (2014) Curr Protoc Mol Biol 105: 15.14