The LAMP method
This method relies on auto-cycling strand displacement DNA synthesis that is performed by a DNA polymerase with high strand displacement activity and a set of two specially designed inner and two outer primers. In the initial steps of the LAMP reaction, all four primers are used, but later during the cycling reaction only the inner primers are used for strand displacement DNA synthesis. The inner primers are called the forward inner primer (FIP) and the backward inner primer (BIP), respectively, and each contains two distinct sequences corresponding to the sense and antisense sequences of the target DNA, one for priming in the first stage and the other for self-priming in later stages. For ease of explanation, the sequences (typically 23–24 nt) inside both ends of the target region for amplification in a DNA are designated F2c and B2, respectively (Fig. ). Two inner sequences (typically 23–24 nt) 40 nt from the ends of F2c and B2 are designated F1c and B1 and two sequences (17–21 nt) outside the ends of F2c and B2 are designated F3c and B3. Given this structure, the sequences of FIP and BIP were designed as follows. FIP contains F1c, a TTTT spacer and the sequence (F2) complementary to F2c. BIP contains the sequence (B1c) complementary to B1, a TTTT spacer and B2. The two outer primers consist of B3 and the sequence (F3) complementary to F3c, respectively. A DNA sample containing the target sequence and the four primers is heat denatured and rapidly cooled on ice. The LAMP reaction is then initiated by addition of the Bst DNA polymerase large fragment and carried out at 65°C for 1 h.
Figure 1 Schematic representation of the mechanism of LAMP. (A) Steps in the LAMP reaction. This figure shows the process that starts from primer FIP. However, it should be remembered that DNA synthesis can also begin from primer BIP. (B) Schematic presentation (more ...)
The mechanism and expected reaction steps of LAMP are illustrated in Figure . Inner primer FIP hybridizes to F2c in the target DNA and initiates complementary strand synthesis (Fig. A). Outer primer F3, which is a few bases shorter and lower in concentration than FIP, slowly hybridizes to F3c in the target DNA and initiates strand displacement DNA synthesis, releasing a FIP-linked complementary strand, which can form a looped out structure at one end (structure 4). This single-stranded DNA serves as template for BIP-initiated DNA synthesis and subsequent B3-primed strand displacement DNA synthesis, leading to the production of a dumb-bell form DNA (structure 6), which is quickly converted to a stem–loop DNA by self-primed DNA synthesis (structure 7). This stem–loop DNA then serves as the starting material for LAMP cycling, the second stage of the LAMP reaction.
To initiate LAMP cycling, FIP hybridizes to the loop in the stem–loop DNA (structure 7) and primes strand displacement DNA synthesis, generating as an intermediate one gapped stem–loop DNA with an additional inverted copy of the target sequence in the stem and a loop formed at the opposite end via the BIP sequence (structure 8). Subsequent self-primed strand displacement DNA synthesis yields one complementary structure of the original stem–loop DNA (structure 10) and one gap repaired stem–loop DNA with a stem elongated to twice as long (double copies of the target sequence) and a loop at the opposite end (structure 9). Both these products then serve as template for a BIP-primed strand displacement reaction in the subsequent cycles, a part of which is designated the elongation and recycling step, illustrated in the right half of Figure A. Thus, in LAMP the target sequence is amplified 3-fold every half cycle.
The final products are a mixture of stem–loop DNAs with various stem lengths and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target sequence in the same strand (Fig. , structures 16–18). The structures of the cycling intermediate and final products are schematically illustrated in Figure B in linearized DNA form.
The use of four primers (recognition of six distinct sequences) in the initial steps of LAMP and two primers (recognition of four distinct sequences) during the subsequent steps ensures high specificity for target amplification. Moreover, in LAMP four primers (six distinct recognition sequences) are simultaneously used to initiate DNA synthesis from the original unamplified DNA to generate a stem–loop DNA for subsequent LAMP cycling, during which the target is recognized by four sequences. Therefore, target selectivity is expected to be higher than those obtained in PCR and SDA.
LAMP amplification of M13 DNA as a model
In order to demonstrate the mechanism, the efficiency, the specificity and the ease of use of LAMP, we chose M13mp18 DNA as a model target DNA and prepared four primers that met the LAMP requirements (Fig. A). The reaction was carried out at 65°C for 1 h and the products were separated by agarose gel electrophoresis and identified by restriction enzyme digestion and Southern blot hybridization with appropriate probes (Fig. A and B). The LAMP reaction produced many bands of different sizes from ~300 bp to the loading well (Fig. A, lane 4). Production of the bands depended on the presence of the inner primers, the template and DNA polymerase. When the products were analyzed by alkaline agarose gel electrophoresis, smeared DNA between bands and at the well (shown in Fig. A, lane 4) was shifted to bands of <10 kb (Fig. E). Thus, we attributed the slow migrating DNA and the DNA in the loading well to replicating intermediates containing single-stranded loops, as shown in Figure A.
Figure 2 (A) Nucleotide sequence of M13mp18 used for designing the inner and outer primers. The nucleotide sequence of the sense strand of M13mp18 DNA is shown. DNA sequences used for primer design are shown by heavy lines. Probe sequences used for Southern blot (more ...)
Figure 3 Restriction analysis and Southern blot hybridization of the amplified M13mp18 DNA. (A) Electrophoretic analysis of the LAMP amplified M13mp18 product. Six hundred copies of M13mp18 DNA were amplified by LAMP with the specific primers designed on the (more ...)
To confirm the structure, the amplified products were digested with several restriction endonucleases and their sizes analyzed by electrophoresis. BamHI cuts B1, PstI cuts between F1c and B1, and PvuII cuts between F1c and F2c (Fig. A). Consequently, if the amplification products had exactly the structures shown in Figure , the products would be fragmented to 101 and 230 bp fragments by BamHI digestion, 137 and 194 bp fragments by PstI digestion, and 237, 315 and 347 bp fragments by PvuII digestion. As shown in Figure A, the sizes of the fragments generated were approximately 100 and 230 bp for BamHI, 140 and 200 bp for PstI, and 240, 320 and 350 bp for PvuII digestion (Fig. A, lanes 5–7), in good agreement with the predicted sizes (Fig. B). To further confirm their structure, the restriction digests were analyzed by Southern blot hybridization using the sequence complementary to the inner region between F1c and B1 (M13-281), the sequence complementary to the inner region between F1c and F2c (M13-333), and the BIP primer itself as hybridization probes. M13-281 and M13-333 hybridized to the 194 bp PstI product, but not to the 137 bp fragment (Fig. B and C, lane 6). In contrast, BIP hybridized to the 137 bp, but not the 194 bp fragment (Fig. D, lane 6). Similarly, Southern blot results of the PvuII and BamHI digests perfectly agreed with the conclusion that the amplified DNA originated from target M13 DNA (Fig. B–D, lanes 5 and 7). The structures of the amplified products were also confirmed by cloning and sequencing. Several bands shown in Figure A, lane 4, were isolated and cloned after digestion with mung bean nuclease. The sequences of the cloned DNAs perfectly agreed with the expected nucleotide sequences (data not shown).
Optimized conditions for LAMP
Since hybridization of the four primers to the target DNA in the initial step was critical for the efficiency of LAMP, the sequences and sizes of the primers were chosen so that their melting temperatures (Tm) fell within certain ranges. The F2 and B2 sequences in FIP and BIP were chosen such that their Tm values fell between 60 and 65°C, the optimal temperature for Bst polymerase. The Tm values of F1c and B1c were set slightly higher than those of F2 and B2 in order that a looped out structure formed immediately after release of the single-stranded DNA from the template. Furthermore, the Tm values of the outer primers (F3 and B3) were set lower than those of F2 and B2 in order to ensure that synthesis occurred earlier from the inner primers than from the outer primers. In addition, the outer primers were used at 1/4–1/10 the concentration of the inner primers.
The formation of a stem–loop DNA (sees Fig. , structure 7) from a dumb-bell structure (structure 6) is critical for LAMP cycling. We examined the effect of various sizes of loop between F2c (B2c) and F1c (B1c) on amplification efficiencies and found that a loop of 40 bases or longer gave the best results (data not shown).
The efficiency of LAMP depends on the size of the target DNA because one rate limiting step for amplification in this method is strand displacement DNA synthesis. We tested various sizes of target DNA and found that the best results could be obtained with 130 to 200 bp DNAs. DNA of more than 500 bp amplified, but very poorly. Therefore, the size of target DNA should be set to less than 300 bp, including F2 and B2.
DNA polymerase is another critical factor for efficient amplification. The best amplification was obtained with Bst polymerase or BcaBEST DNA polymerase (TaKaRa) for less than 10–23 mole target DNA. Z-Taq DNA polymerase (TaKaRa) was less efficient under the current conditions, but might be useful when polymerase has to be added before heat denaturation of the target DNA, because it is thermostable.
Chemicals destabilizing the DNA helix were found to markedly elevate amplification efficiencies in LAMP. The presence of 0.5–1.5 M betaine (N
-trimethylglycine) or l
-proline, which reduce base stacking (9
), stimulated not only the overall rate of the reaction, but also increased target selectivity with a significant reduction in amplification of irrelevant sequences.
Sensitivity of LAMP
LAMP is highly sensitive and able to detect DNA at as few as six copies in the reaction mixture. As shown in Figure A, in a 45 min LAMP reaction six copies of the HBV target were amplified to a detectable level. As expected, use of inner primers that do not form a looped out structure led to no amplification (Fig. B, lanes 5–8). In the absence of one of the outer primers no significant amplification occurred with 60 copies of the HBV target (Fig. B, lanes 2–4), indicating a strict requirement for recognition of six distinct sequences in the target DNA in LAMP.
Figure 4 Sensitivity of LAMP. (A) Time course of the LAMP reaction with various amounts of HBV DNA. Various numbers of copies of HBV DNA were amplified by LAMP. At various times, the reaction was terminated and the amounts of products quantified by measuring (more ...)
LAMP seems less prone to the presence of irrelevant DNA than PCR. The presence of 100 ng of human genomic DNA in the LAMP reaction for six copies of HBV target neither significantly adversely affected the amplification efficiency nor generated significant background (Fig. C). Single PCR, which was performed under the same conditions as for LAMP, failed to amplify six copies of HBV in the absence and 60 copies in the presence of 10–100 ng of the genomic DNA, though nested PCR overcame this problem (Fig. D).
LAMP for a RNA target
LAMP is also applicable to RNA upon use of reverse transcriptase (RTase) together with DNA polymerase (12
). This method (reverse transcription-coupled LAMP) easily detected prostate-specific antigen (PSA) mRNA in one PSA-expressing LNCaP cell mixed with 1 000 000 PSA-negative K562 cells (13
; Fig. , lane 6). This amplification depended on both RTase and Bst
polymerase (Fig. , lanes 2 and 4) and the product was authenticated by Sau
3AI digestion (Fig. , lanes 8 and 9).
Figure 5 Detection of PSA mRNA by reverse transcription-coupled LAMP (RT-LAMP). Various numbers of LNCaP cells were mixed with 106 PSA-non-producing K562 cells and total RNA was extracted. RT-LAMP was carried out in the same reaction mixture as for M13mp18 DNA (more ...)
Advantages of LAMP
(i) LAMP amplifies DNA with high efficiency under isothermal conditions without a significant influence of the co-presence of non-target DNA. Its detection limit is a few copies, being comparable to that of PCR. (ii) The products are a mixture of stem–loop DNAs with various sizes of stem and cauliflower-like structures with multiple loops induced by annealing between alternately inverted repeats of the target sequence in the same strand, the latter of which would enable their simple, easy and selective detection, such as via mechanisms similar to multivalent antigen–antibody interactions. (iii) LAMP is highly specific for the target sequence. This is attributable to recognition of the target sequence by six independent sequences in the initial stage and by four independent sequences during the later stages of the LAMP reaction. This partly alleviates the general problem of backgrounds associated with all nucleic acid amplification methods. (iv) LAMP is simple and easy to perform once the appropriate primers are prepared, requiring only four primers, a DNA polymerase and a regular laboratory water bath or heat block for reaction. (v) By combination with reverse transcription, LAMP can amplify RNA sequences with high efficiency.