Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products

Journal name:
Nature Protocols
Volume:
3,
Pages:
877–882
Year published:
DOI:
doi:10.1038/nprot.2008.57
Published online
Corrected online

Abstract

As the human genome is decoded and its involvement in diseases is being revealed through postgenome research, increased adoption of genetic testing is expected. Critical to such testing methods is the ease of implementation and comprehensible presentation of amplification results. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, specific and cost-effective nucleic acid amplification method when compared to PCR, nucleic acid sequence-based amplification, self-sustained sequence replication and strand displacement amplification. This protocol details an improved simple visual detection system for the results of the LAMP reaction. In LAMP, a large amount of DNA is synthesized, yielding a large pyrophosphate ion by-product. Pyrophosphate ion combines with divalent metallic ion to form an insoluble salt. Adding manganous ion and calcein, a fluorescent metal indicator, to the reaction solution allows a visualization of substantial alteration of the fluorescence during the one-step amplification reaction, which takes 30–60 min. As the signal recognition is highly sensitive, this system enables visual discrimination of results without costly specialized equipment. This detection method should be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and more.

At a glance

Figures

  1. Principle of loop-mediated isothermal amplification (LAMP) method.
    Figure 1: Principle of loop-mediated isothermal amplification (LAMP) method.

    (a) Primer design of the LAMP reaction. For ease of explanation, six distinct regions are designated on the target DNA, labeled F3, F2, F1, B1c, B2c and B3 from the 5′ end. As c represents a complementary sequence, the F1c sequence is complementary to the F1 sequence. Two inner primers (FIP and BIP) and outer primers (F3 and B3) are used in the LAMP method. FIP (BIP) is a hybrid primer consisting of the F1c (B1c) sequence and the F2 (B2) sequence. (b) Starting structure producing step. DNA synthesis initiated from FIP proceeds as follows. The F2 region anneals to the F2c region on the target DNA and initiates the elongation. DNA amplification proceeds with BIP in a similar manner. The F3 primer anneals to the F3c region on the target DNA, and strand displacement DNA synthesis takes place. The DNA strand elongated from FIP is replaced and released. The released single strand forms a loop structure at its 5′ end (structure 4). DNA synthesis proceeds with the single-strand DNA as the template, and BIP and B3 primer, in the same manner as described earlier, to generate structure 5, which possesses the loop structure at both ends (dumbbell-like structure). (c) Cycling amplification step. Using structure 5 as the template, self-primed DNA synthesis is initiated from the 3′ end F1 region, and the elongation starts from FIP annealing to the single strand of the F2c region in the loop structure. Passing through several steps, structure 7 is generated, which is complementary to structure 5, and structure 5 is produced from structure 8 in a reaction similar to that which led from structures 5–7. Specifically, intermediate structures 7a and 9a and structures 5a and 10a (in the yellow boxes) are produced from structures 6 and 8, respectively. Structures 9a and 10a then form structures 9 and 10, respectively, whereas the displaced strands 7a and 5a form the dumbbell-like structures 7 and 5, respectively. More elongated structures (11, 12) are also produced. (The authors acknowledge S. Subramanian and R.D. Gomez of the University of Maryland, who brought the error to their attention and supplied the corrected version of Figure 1c and the legend.)

  2. Principle of detection using a fluorescent metal indicator (calcein).
    Figure 2: Principle of detection using a fluorescent metal indicator (calcein).

    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.

  3. Detection of the loop-mediated isothermal amplification (LAMP) reaction using fluorescent metal indicator.
    Figure 3: Detection of the loop-mediated isothermal amplification (LAMP) reaction using fluorescent metal indicator.

    (a) Irradiating the tube using a handheld-UV lamp (wavelength: 365 nm) from the bottom. (b) Under daylight. Plus sign denotes positive reaction (with target DNA), minus sign denotes negative reaction (without target DNA).

  4. Fluorescence spectra of the reaction solution after the amplification reaction (excitation at 480 nm).
    Figure 4: Fluorescence spectra of the reaction solution after the amplification reaction (excitation at 480 nm).

    Black line: with target DNA, Mn2+ ion 0 mM; green line: without target DNA, Mn2+ ion 0 mM; red line: with target DNA, Mn2+ ion 0.5 mM; blue line: without target DNA, Mn2+ ion 0.5 mM. All reaction solutions contain 25 μM of calcein.

  5. Analysis of the loop-mediated isothermal amplification (LAMP) reaction products using agarose gel electrophoresis.
    Figure 5: Analysis of the loop-mediated isothermal amplification (LAMP) reaction products using agarose gel electrophoresis.

    Lane 1: with target DNA, Mn2+ ion 0 mM; lane 2: without target DNA, Mn2+ ion 0 mM; lane 3: with target DNA, Mn2+ ion 0.5 mM; lane 4: without target DNA, Mn2+ ion 0.5 mM; lane M: 100-bp DNA ladder used as size maker. Each sample was electrophoresed in 2.5% agarose gel. The samples were same as in Figure 4. The characteristic ladder pattern for LAMP products was observed in lanes 1 and 3.

Change history

Corrected online 12 January 2012
In the version of this article initially published, the legend for Figure 1 contained errors. In the description of panel b, the sentence "The released single strand forms a loop structure at its 3′ end (structure 3)" should have ended with "a loop structure at its 5′ end (structure 4)." In the description of panel c, "Using self-structure as the template..." should have read "Using structure 5 as the template..." The last sentence of the legend ("Structures 9 and 10 are produced from structures 6 and 8, respectively, and more elongated structures (11, 12) are also produced.") has been replaced with the following: "Specifically, intermediate structures 7a and 9a and structures 5a and 10a (in the yellow boxes) are produced from structures 6 and 8, respectively. Structures 9a and 10a then form structures 9 and 10, respectively, whereas the displaced strands 7a and 5a form the dumbbell-like structures 7 and 5, respectively. More elongated structures (11, 12) are also produced." Finally, in panel c of the figure, structure 9 (with downstream structure 11) and structure 10 (with downstream structure 12) were shown in incorrect locations, and additional structures (5a, 7a, 9a, and 10a) were omitted. These errors have been corrected in the HTML and PDF versions of the article. The authors acknowledge S. Subramanian and R.D. Gomez of the University of Maryland, who brought the error to their attention and supplied the corrected version of Figure 1c and legend.

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Affiliations

  1. Eiken Chemical Co., Ltd., Biochemical Research Laboratory, 1381-3 Shimoishigami, Ohtawara, Tochigi 324-0036, Japan.

    • Norihiro Tomita,
    • Yasuyoshi Mori,
    • Hidetoshi Kanda &
    • Tsugunori Notomi

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