LAMP PCR

ABBREVIATION

LAMP

INTRODUCTION• Solely developed by Eiken Chemical Co.,

Ltd.• First reported by Notomi et al in 2000 of

EIKEN Chemical Co. Ltd., Japan. (http://www.eiken.co.jp/en/)

BASIC PRINCIPLE

Procedure of LAMP PCR1- Design of Primers• 4 types of primers based on the 6 distinct regions of the target

gene: the F3c, F2c and F1c regions at the 3' side and the B1, B2 and B3 regions at the 5' side

Continued…2- Amplification• Is of two types;A. Non-cycling AmplificationB. Cycling Amplification

A.Non-cycling Amplification:

• Generation of stem loop DNA with dumbbell-shaped structure at both ends.

B. Cycling Amplification:

• Dumbbell-shaped DNA is quickly amplified by the use of loop primers.

NON-CYCLING AMPLIFICATION

Step-1• One of the LAMP primers anneal to the complimentary

sequence of double stranded target DNA.• Initiates DNA synthesis using the DNA polymerase with

strand displacement activity, displacing and releasing a single stranded DNA.

• Unlike PCR, no need for heat denaturation of ds DNA.

Continued…Step-2• Through the strand displacement

activity of DNA polymerase, a DNA strand complementary to the template DNA is synthesized, starting from the 3' end of the F2 region of the FIP.

Step-3• The F3 Primer anneals to the F3c

region, outside of FIP, on the target DNA and

• Initiates strand displacement DNA synthesis, releasing the FIP-linked complementary strand.

Continued…Step-4• A double strand is formed from

the DNA strand synthesized from the F3 Primer and the template DNA strand.

Step-5• The FIP-linked complementary

strand is released as a single strand because of the displacement by the DNA strand synthesized from the F3 Primer.

• Released single strand forms a stem-loop structure at the 5' end because of the complementary F1c and F1 regions.

Continued…Step-6• BIP-initiated DNA synthesis and

subsequent B3-primed strand displacement DNA synthesis.

Step-7• Double stranded DNA is produced

through the processes described in Step-6.

Step-8• The BIP-linked complementary

strand and forms a structure with stem-loops dumbbell structure at each end, serving as the starting structure for LAMP cycling.

CYCLING AMPLIFICTAION

Continued…3- Detection

i. Visual Detection

• Turbidity - Magnesium pyrophosphate

• Fluorescence – Calcein

ii. Gel Electrophoresis

• Lane 1 and 3 has target DNA.• Lane 2 and 4 has non-target DNA.• Lane M has DNA Ladder.

LAMP vs. PCR

• Isothermal Reaction.• Isothermal Temperature (60-

65⁰C).

• Doesn’t require expensive thermocycler.

• Detection limit is greater.• Amplification specificity is

higher as uses 4/6 oligonucleotides.

• Visualization of DNA could be done through eyes, gel electrophoresis and turbidimeter.

• Cyclic Reaction.• Variable Temperature.

Denaturation (95⁰C)Annealing (50-60⁰C)Polymerization (72⁰C)

• Require thermocycler.

• Detection limit is lower.• Amplification specificity is

lower than that of LAMP.

• Visualization of DNA is done through gel electrophoresis.

Loop-mediated Isothermal Amplification - LAMP

Polymerase Chain Reaction - PCR

• Could be done using crude DNA samples.

• Loop primers accelerate reaction rate.

• Need pure DNA samples for amplification.

• No loop primer.

LAMP vs. PCRLoop-mediated Isothermal

Amplification - LAMPPolymerase Chain Reaction -

PCR

APPLICATIONS OF LAMP

CONCLUSION• LAMP is highly sensitive and specific DNA/RNA

amplification technique.• It works on isothermal conditions and doesn't require

expensive operational machinery.• It is simple, cost effective technique.• LAMP is an innovation molecular diagnostic field and

can be used for the diagnosis of infectious diseases, food inspection, environmental testing and so on.

• Innovations in biotechnology that combine molecular biology, microfabrication and bioinformatics are moving nucleic acid technologies from futuristic possibilities to common laboratory techniques and modes for diagnoses.