 |
 |


 |
 |
The principle of LAMP method |


 |
When
the target gene (DNA template as example) and the reagents are incubated
at a constant temperature between 60-65°C, the following reaction
steps proceed: |
 |
 |
STEP1
As 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 DNA polymerase with strand displacement
activity, displacing and releasing a single stranded DNA. With the
LAMP method, unlike with PCR, there is no need for heat denaturation
of the double stranded DNA into a single strand. The following amplification
mechanism explains from when the FIP anneals to such released single
stranded template DNA. |
 |
 |
 |
 |
 |
|
 |
 |
STEP2
Through the activity of DNA polymerase with strand displacement
activity, a DNA strand complementary to the template DNA is synthesized,
starting from the 3' end of the F2 region of the FIP. |
 |
 |
|
 |
 |
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
because of the displacement by the DNA strand synthesized from the
F3 Primer. Then, this released single strand forms a stem-loop structure
at the 5' end because of the complementary F1c and F1 regions. |
 |
 |
|
 |
 |
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.
The BIP anneals to the DNA strand produced in Step (5). Starting
from the 3' end of the BIP, synthesis of complementary DNA takes
place. Through this process, the DNA reverts from a loop structure
into a linear structure. The B3 Primer anneals to the outside of
the BIP and then, through the activity of the DNA polymerase and
starting at the 3' end, the DNA synthesized from the BIP is displaced
and released as a single strand before DNA synthesis from the B3
Primer. |
 |
 |
 |
 |
 |
|
 |
 |
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). The above
process can be understood as producing the starting structure for
LAMP cycling. |
 |
 |
 |
 |
 |
|
 |
 |
 |
 |
Basic principle (8) - (11) (Cycling amplification step) |
 |
|
 |

 |
A
dumbbell-like DNA structure is quickly converted into a stem-loop
DNA by self-primed DNA synthesis. FIP anneals to the single stranded
region in the stem-loop DNA and primes strand displacement DNA synthesis,
releasing the previously synthesized strand. This released single
strand forms a stem-loop structure at the 3' end because of complementary
B1c and B1 regions. Then, starting from the 3' end of the B1 region,
DNA synthesis starts using self-structure as a template, and releases
FIP-linked complementary strand (Step (9)). The released single
strand then forms a dumbbell-like structure as both ends have complementary
F1 - F1c and B1c - B1 regions, respectively (Step (11)). This structure
is the 'turn over' structure of the structure formed in Step (8).
Similar to the Steps from (8) to (11), structure in Step (11) leads
to self-primed DNA synthesis starting from the 3' end of the B1
region. Furthermore, BIP anneals to the B2c region and primes strand
displacement DNA synthesis, releasing the B1-primed DNA strand.
Accordingly, similar structures to Steps (9) and (10) as well as
the same structure as Step (8) are produced. With the structure
produced in Step (10), the BIP anneals to the single strand B2c
region, and DNA synthesis continues by displacing double stranded
DNA sequence. As a result of this process, various sized structures
consisting of alternately inverted repeats of the target sequence
on the same strand are formed.
|
 |


|
 |
 |
|
 |