New England Biolabs has some very valuable resources to understand what LAMP is, how it works and how to design primers for this purpose. You will find these informations here:

https://international.neb.com/applications/dna-amplification-pcr-and-qpcr/isothermal-amplification/loop-mediated-isothermal-amplification-lamp

Best regards,

John

(P.S. I have no affiliation with NEB)

Abdulla Abdulla The Loop-Mediated Isothermal Amplification (LAMP) technique is a powerful nucleic acid amplification method that allows for rapid and highly specific amplification of target DNA or RNA sequences. LAMP is particularly advantageous because it operates under isothermal conditions, eliminating the need for thermal cycling typically required in traditional PCR methods.

The LAMP reaction relies on a set of specially designed primers that recognize multiple regions within the target sequence. These primers consist of two inner primers (forward inner primer and reverse inner primer) and two outer primers (forward outer primer and reverse outer primer). The presence of multiple primers facilitates the initiation of DNA synthesis from multiple sites on the target sequence, leading to highly efficient and specific amplification.

The LAMP reaction mixture contains a DNA polymerase with strand displacement activity, which allows for continuous DNA synthesis and displacement of the newly synthesized DNA strands from the template. This strand displacement activity is a crucial feature of LAMP, as it enables the amplification process to proceed in a loop-like manner, resulting in the accumulation of a large amount of target DNA.

The LAMP reaction is typically performed at a constant temperature around 60-65°C, which is maintained throughout the amplification process. This isothermal condition is achieved by using a thermostable DNA polymerase, such as Bst DNA polymerase, which exhibits robust activity at elevated temperatures.

During the LAMP reaction, the target DNA or RNA is amplified through a series of steps. Initially, the primers bind to their complementary sequences on the target DNA, forming stem-loop structures. The stem-loop structures serve as templates for the synthesis of new DNA strands.

The amplification process involves several key steps, including:

1. Strand displacement: The DNA polymerase initiates synthesis at the 3' end of the inner forward primer, displacing the original forward inner primer strand.

2. DNA synthesis: The displaced forward inner primer strand hybridizes with the outer forward primer, allowing DNA synthesis to continue along the target DNA sequence.

3. Strand displacement and DNA synthesis: The newly synthesized DNA strand displaces the reverse inner primer strand, allowing for continuous DNA synthesis in the reverse direction.

4. Amplification and loop formation: The displaced reverse inner primer strand hybridizes with the reverse outer primer, initiating DNA synthesis in the forward direction. This creates a loop structure that serves as a template for further amplification.

The amplification process continues in a loop-like manner, with continuous displacement and synthesis of DNA strands. This results in a rapid and exponential increase in the target DNA or RNA, leading to a significant amplification within a relatively short time.

The amplified products of LAMP can be detected using various methods. Visual inspection can be used in some cases, where the presence of amplified DNA causes a color change or turbidity. Fluorescent dyes can also be incorporated into the reaction, and fluorescence-based assays can be employed for real-time monitoring of the amplification process. Gel electrophoresis can be used to visualize the amplified products by separating them based on size.

The LAMP technique has a wide range of applications in various fields, including clinical diagnostics, infectious disease testing, food safety analysis, agricultural research, environmental monitoring, and point-of-care testing. Its high specificity, sensitivity, and simplicity make it a valuable tool for rapid and reliable nucleic acid amplification.

Hi Abdulla,

LAMP (Loop-Mediated Isothermal Amplification) PCR is a nucleic acid amplification technique used to rapidly and specifically amplify DNA or RNA sequences. It is a powerful molecular biology tool that has found applications in various fields, including medical diagnostics, agricultural testing, and environmental monitoring.

Unlike traditional PCR methods that involve temperature cycling, LAMP PCR operates at a constant temperature, typically around 60-65 degrees Celsius. This isothermal amplification allows for simple and cost-effective setups, as specialized thermal cycling equipment is not required.

LAMP PCR utilizes a set of four to six primers that specifically target multiple regions of the DNA or RNA sequence of interest. These primers consist of two outer primers (forward and reverse), two inner primers (forward and reverse), and optionally, two loop primers. The design of these primers is crucial for the amplification process.

The LAMP reaction involves strand displacement, where the target DNA or RNA sequence is amplified through a series of DNA synthesis and displacement steps. The process creates stem-loop DNA structures with multiple regions that serve as templates for continuous amplification. The LAMP PCR reaction proceeds through a series of steps, including denaturation, annealing, extension, and strand displacement. The reaction is driven by DNA polymerase with high strand displacement activity, such as Bst DNA polymerase. The continuous amplification occurs in a rapid and exponential manner, leading to a significant increase in the target sequence.

LAMP PCR has a broad range of applications due to its simplicity, rapidity, and high sensitivity. It has been used for the detection of various pathogens, including viruses, bacteria, and parasites. Additionally, LAMP PCR can be used for genetic testing, food safety analysis, environmental monitoring, and point-of-care diagnostics.

Overall, the LAMP PCR technique offers a powerful and versatile method for the amplification and detection of specific DNA or RNA sequences under isothermal conditions, making it a valuable tool in molecular biology and diagnostic.

Best regards