PCR Type, LAMP PCR, New PCR Technique, Polymerase Chain Reaction LAMP,

1. LAMP PCR

2. ABBREVIATION
LAMPLAMP

3. 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/)

4. BASIC PRINCIPLE

6. Procedure of LAMP PCR
1- 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

7. Continued…
2- Amplification
• Is of two types;
A. Non-cycling Amplification
B. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. CYCLING AMPLIFICTAION

14. 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.

15. LAMP vs. PCR
• Isothermal Reaction.
• Isothermal Temperature (60-
65 C).⁰
• 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 (95 C)⁰
Annealing (50-60 C)⁰
Polymerization (72 C)⁰
• 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

16. • Could be done using crude
DNA samples.
• Loop primers accelerate
reaction rate.
• Need pure DNA samples
for amplification.
• No loop primer.
LAMP vs. PCR
Loop-mediated Isothermal
Amplification - LAMP
Polymerase Chain Reaction - PCR

17. APPLICATIONS OF LAMP

18. 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.