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| The principle of LAMP method |
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Design
4 types of primers (described in detail below) based on the following
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.
| FIP |
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Forward Inner
Primer (FIP) consists of the F2 region (at the 3' end) that
is complementary to the F2c region, and the same sequence
as the F1c region at the 5' end. |
| F3 Primer |
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Forward Outer
Primer consists of the F3 region that is complementary to
the F3c region. |
| BIP
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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 Primer |
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Backward
Outer Primer consists of the B3 region that is complementary
to the B3c region. |
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Main points of primer design |
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Proper
primer design is crucial for performing LAMP amplification.The above
primer regions can be determined by using the PrimerExplore (a special
software to design LAMP primers) after considering the base composition,
GC contents and the formation of secondary structures. Tm value
can be obtained by Nearest Neighbor method.
The following is the main points of primer design.
| 1.Distance
between primer regions |
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The distance between 5' end of F2 and
B2 is considered to be 120-180bp, and the distance between
F2 and F3 as well as B2 and B3 is 0-20bp. |
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The distance for loop forming regions
(5' of F2 to 3' of F1, 5' of B2 to 3' of B1) is 40-60bp. |
| 2.Tm value
for primer regions |
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About 60-65°C in the case of GC rich
and Normal, about 55-60°C for AT rich. |
| 3.The stability
of primer end |
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The dG calculated on 6bp from the following
end regions should be less than -4kcal/mol, 5' end of F1c/B1c
and 3' end of F2/B2 as well as F3/B3. |
| 4.GC contents |
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About 50-60% in the case of GC rich
and Normal, about 40-50% for AT rich. |
| 5.Secondary
structure |
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Primers should be designed so as not
to easily form secondary structures. 3' end sequence should
not be AT rich or complementary to other primers. |
| 6.Others |
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If the restriction enzyme sites exist
on the target sequence, except the primer regions, they can
be used to confirm the amplified products. |
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| Click here to start primer design.
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We have received
various inquires about primer design, in some cases,
negative samples were amplified, or non-specific reactions
were observed. Reasons for these cases might include:
(1) contamination has occurred. (2) primer dimmers were
formed and from this the amplification proceeded. In
the case of (1), changing to new reagents, instruments
and equipments can help to avoid contamination. However,
in case (2), as primers were not designed properly,
re-design of the primers is needed. |
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