Loop-mediated isothermal amplification of DNA

Abstract

We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.

Figures

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 of the structure of LAMP products in a linearized DNA form. B+, B–, F+ and F– stand for the DNA structures shown in the boxes on the left. +, the target sequence flanked by B1 and F1c; –, the complementary sequence.

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 hybridization are indicated by dotted lines and the restriction sites for BamHI, PstI and PvuII are indicated by boxes. Numbers at the left end correspond to the positions in M13mp18 (GenBank accession no. X02513). (B) Schematic representation of the anticipated structure of the amplified product. B+, B–, F+ and F– in the first row are as in Figure 1B. The second row indicates the probes used for Southern blot hybridization. The restriction sites for PstI, PvuII and BamHI are shown as lines and the sizes of the restriction fragments are in the boxes.

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 sequences shown in Figure 2 and run on a 2% agarose gel followed by SYBR Green I staining. Lane M, 100 bp ladder used as size marker (New England Biolabs); lane 1, M13mpl8 DNA digested with PvuII; lane 2, LAMP without Bst DNA polymerase; lane 3, LAMP without target M13 DNA; lane 4, complete LAMP; lanes 5–7, complete LAMP products after digestion with BamHI, PstI and PvuII, respectively (one fifth of the digests were loaded). (B–D) Southern blot analysis of the LAMP products. The 2% agarose gel shown in (A) was used for Southern blot hybridization with M13-281 DNA (B), M13-333 DNA (C) and M13BIP (D) as probes. (E) Alkaline agarose gel electrophoresis of the LAMP products. Lane m, λ DNA HindIII digests; lane 4, the same sample as in (A).

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 fluorescence intensity of SYBR Green I. (B) Requirements for primers in the LAMP reaction. Sixty copies of HBV DNA were amplified by LAMP with omission of one or two of the primers. The products were electrophoresed in 2% agarose gels and stained. –, the corresponding primer was omitted from the reaction; B2 and F2, BIP and FIP were replaced by B2 and F2, respectively, which do not contain B1c and F1c and, therefore, are unable to form the looped out structure. (C) The effect of the presence of genomic DNA on sensitivity. Various numbers of copies of HBV DNA were amplified at 60°C for 60 min in the absence or presence of 100 ng of human genomic DNA and the products separated by gel electrophoresis. Lane M, 100 bp ladder size markers (TaKaRa); lanes 1–4, LAMP carried out in the absence of human genomic DNA; lanes 5–8, LAMP carried out in the presence of 100 ng genomic DNA; lanes 1 and 5, LAMP without HBV DNA; lanes 2 and 6, with six copies; lanes 3 and 7, with 60 copies; lanes 4 and 8, with 600 copies of HBV DNA. Lanes 9 and 10 are EarI digests (1/5 vol) of the same amplified DNAs as in lanes 2 and 6, respectively. (D) Nested PCR of HBV DNA under similar conditions. Single and nested PCR reactions were performed in a 50 µl reaction mixture containing 2.5 U AmpliTaq Gold (PE Biosystems), 0.2 µM each primer (first PCR, HBVB2/HBVF2 or HBVB1/HBVF1; second PCR, HBVB1/HBVF1), 1× GeneAmp PCR buffer (10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin) and 0.2 mM dNTPs. The sequences of the primers used were 5′-CCAACCTCTTGTCCTCCAA-3′ for HBVB2, 5′-GACAAACGGGCAACATACCTT-3′ for HBVF2, 5′-GGATGTGTCTGCGGCGTTTTATC-3′ for HBVB1 and 5′-AGAAGATGAGGCATAGCAGCAGG-3′ for HBVF1. Both the first and second round nested PCRs were carried out as follows: preincubation at 95°C for 10 min; 40 cycles each of 30 s at 95°C, 30 s at 60°C and 1 min at 72°C. One microliter of the first PCR products (HBVB2/HBVF2) was subjected to second PCR. An aliquot of 10 µl of the reaction products was analyzed by 4% agarose gel (0.5× TBE) electrophoresis followed by staining with SYBR Green I.

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 amplification except that 1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M betaine, 5 mM DTT, 16 U Bst polymerase, 100 U ReverTra Ace (Toyobo) and 5 µg of extracted RNA were used. All the above components were mixed at once on ice and were incubated at 65°C for 45 min. The products were electrophoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-LAMP carried out in the presence and absence of Bst DNA polymerase or ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in lanes 6 and 7, respectively, but digested with Sau3AI; lane M, 100 bp ladder (New England Biolabs).