2. Nucleic acid amplification is one of the most valuable tools in
virtually all life science fields
PCR; polymerase chain reaction
NASBA; nucleic acid sequence-based amplification
SMART; signal mediated amplification of RNA technology
SDA; strand displacement amplification
RCA; rolling circle amplification
LAMP; loop-mediated isothermal amplification of DNA
HDA; helicase-dependent amplification
SPIA; single primer isothermal amplification
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7. Loop mediated isothermal amplification (LAMP) is a
single tube technique for the amplification of DNA offering
rapid, accurate, and cost-effective diagnosis of infectious
diseases
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8. Characteristics
─ no need for a step to denature double stranded into a single stranded form.
─ The whole amplification reaction takes place continuously under isothermal
conditions (water bath).
─ The amplification efficiency is extremely high. the amount of DNA ( 109-1010
times in 15-60 minutes) produced in LAMP is considerably higher than PCR
based amplification
─ The target sequence is amplified at a constant temperature of 60 - 65 C
using 4 primers to recognize 6 distinct regions, and a polymerase with
high strand displacement & replication activity
─ The total cost can be reduced, as it dose not require special reagents or
sophisticated equipment's (Thermal cycler).
─ Amplification also can be done with RNA templates following the same
procedure, simply through the addition of reverse transcriptase.
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9. Design of primers
Design 4 types of primers based on the following 6 distinct regions of the target
gene:
At the 3' side: F3c, F2c and F1c regions
F3c F2c F1c Target DNA
3' 5'
5' 3'
F3 F2 F1
At the 5' side: B1, B2 and B3 regions
Target DNA B1 B2 B3
3' 5'
5' 3'
B1c B2c B3c
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10. T CA
C G T
FIP Forward Inner Primer (FIP) consists of the F2 A T G
region (at the 3' end) that is complementary to
the F2c region, and the same sequence as the GA C
F1c region at the 5' end.
F3 Forward Outer Primer consists of the F3 region
Primer that is complementary to the F3c region.
BIP Backward Inner Primer (BIP) consists of the B2
region (at the 3' end) that is complementary to
the B2c region, and the same sequence as the
B1c region at the 5' end.
B3 Backward Outer Primer consists of the B3
Primer region that is complementary to the B3c region.
http://loopamp.eiken.co.jp/e/lamp/primer.html
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11. Reaction mixture for LAMP
0.8 µM each BIP&FIP
0.2 µM each F3&B3
400 µM dNTPs
1M betaine
25 µl 20 mM Tris-HCL
Reaction
Mixture 10 mM KCL 60-65 C for 1 h
10 mM (NH4) 2 SO4
4 mM MgSO4
0.1% Triton X-100
Bst DNA polymerase
Target DNA
Terminate by 80-
90 C for 5-10 m
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12. Betaine
Reduce base stacking,
Stimulated the overwall rate of the reaction
Increased target selectivity with a significant
reduction of irrelevent sequences
N,N,N-trimethylglycine
Bst DNA polymerase
Bst polymerase is a thermostable DNA Pol I that was
isolated in 1968 (Stenesh 1968) from the thermophilic
bacterium Bacillus stearothermophilus (Bst), which
proliferates between 39 and 70 C. Bst Pol I is active at
an optimal temperature of 65 C, and is inactivated after
15 min incubation at 75 C.
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13. Mechanism or Basic principle
1. Initial step: starting material producing step
Double stranded DNA is in the condition of dynamic
equilibrium at the temperature around 65 C, One of
the LAMP primers can anneal to the complimentary
sequence of double stranded target DNA, then
initiates DNA synthesis using the Bst DNA
polymerase with strand displacement
activity, displacing and releasing a single stranded
DNA.
STEP1
Inner primer FIP anneal to F2c in the target DNA
STEP2
Through the activity of DNA polymerase with strand
displacement activity,initates complementary strand
synthesis
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14. STEP3
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.
STEP4
A double strand is formed from the DNA strand
synthesized from the F3 Primer and the template
DNA strand.
STEP5
The FIP-linked complementary strand is released
as a single strand which forms a stem-loop
structure at the 5' end because of the
complementary F1c and F1 regions
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15. STEP6
This single strand DNA in Step (5) serves as a
template for BIP-initiated DNA synthesis and
subsequent B3-primed strand displacement DNA
synthesis.
STEP7
Double stranded DNA is produced through the
processes described in Step (6).
STEP8
The BIP-linked complementary strand displaced
in Step (6) forms a structure with stem-loops at
each end, which looks like a dumbbell structure.
This structure serves as the starting structure for
the amplification cycle in the LAMP method
(LAMP cycling).
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16. 2. Cycling amplification step
FIP anneals to stem- loop
primes strand displacement
Generate one stem-loop and
additional inverted copy of
target sequence in the stem
where loop form at opposite
end via BIP
Subsequent self primed strand
displacement DNA synthesis
yields one complementary
structure of original stem-loop
Thus ,in LAMP the target
sequence is amplified 3-fold
every half cycle
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17. 3. Elongation and recycling step
The final products are mixture of stem-
loop DNAs with stem lengths and
cauliflower - like structures with
multiple copies
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18. Mechanism or Basic principle- Video
http://www.youtube.com/watch?v=5Wi-kkSFy48 http://loopamp.eiken.co.jp/e/lamp/anim.html
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19. Use of loop primer
The Loop Primers containing sequences complementary to the
single stranded loop region (either between the B1 and B2
regions, or between the F1 and F2 regions) on the 5' end of the
dumbbell-like structure, provide an increased number of
starting points for DNA synthesis for the LAMP method.
The time required for amplification with Loop Primers is
one-third to one-half of that without Loop Primer. With
the use of Loop Primers, amplification can be achieved
within 30 minutes.
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20. Analysis of product
1. Gel elctrophoresis: 2-3% agarose gel stained with EB / SYBR green or
Calcein
2. Naked eye: Photometry for turbidity caused by increasing quantity of
Magnesium pyrophosphate in solution with or without addition of
SYBR green or manganese loaded calcein
3. Real time: either by measuring the turbidity or the signals from DNA
produced via fluorescent dyes that intercalate or directly label the
DNA, and in turn can be correlated to the number of copies initially
present. So, LAMP can also be quantitative.
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22. A) The positive PCR reactions show a 273 bp amplicon while
A) LAMP produces a characteristic ladder of multiple bands on an agarose gel
indicating stem-loop DNA with inverted repeats of the target sequence.
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23. Visual detection of sample by LAMP from
positive and negative samples.
(1) Naked eye detection without SYBR green;
(2) naked eye detection with SYBR green;
(3) detection under an UV trans-illuminator
using SYBR green;
(4) detection under a hand-held black light
using SYBR green.
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24. Uses and benefits
Simple screening assay in the field or at the point of care by clinicians
Low cost
Amplified at a constant temperature of 60 - 65 °C
Easy visualization by the naked eye
The reaction can be followed in real-time
This technology has been developed into commercially available
detection kits for a variety of pathogens including
bacteria, parasite, fungus and viruses
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28. Thank you so much for
your kind attention
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29. In the DNA amplification process by DNA polymerase, pyrophosphate ions are produced as a
by-product from the reaction substrate deoxyribonucleotide triphosphates (dNTPs). The calcein
in the reaction mixture initially combines with manganous ion (Mn2+) so as to remain quenched.
When the amplification reaction proceeds, manganous ion is deprived of calcein by the
generated pyrophosphate ion (P2O74- ), which results in the emission of fluorescence. And the
free calcein is apt to combine with magnesium ion (Mg2+) in the reaction mixture, so that it
strengthens the fluorescence emission.
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