Loop-mediated isothermal amplification of DNA

Abstract

We have developed a novel method, termed loopmediated 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.

Intraoperative molecular assay for sentinel lymph node metastases in early stage breast cancer

“…10 Briefly, after removing extranodal tissue and lipid, a whole lymph node was homogenized with 4 mL of a lysis buffer solution (Lynorhag; Sysmex) and centrifuged at 10,000 Â g at room temperature. A 2 lL sample of the supernatant was analyzed with the RD100i system (Sysmex), an automated gene amplification detection system using a reverse transcription loop-mediated isothermal amplification method, 14 and with the LynoampBC (Sysmex). The degree of amplification was detected via a byproduct of the reaction, pyrophosphate.…”

Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection

“…This technique has been proven to be an accurate, rapid and simple method, which amplifies the target nucleic acid under isothermal conditions (Notomi et al, 2000). Recently, wide applicability of LAMP in the detection of parasitic protozoa such as Babesia, Plasmodium, Leishmania and Trypanosoma in clinical samples has been demonstrated (Ikadai et al, 2004;Poon et al, 2006;Njiru et al, 2008;Takagi et al, 2009;Thekisoe et al, 2007;Laohasinnarong et al, 2011).…”

Cleavage-based signal amplification of RNA

“…Thermal cycling of the PCR technique imposes instrumental constraints, limiting the technique to a laboratory setting, and dual-labelled fluorescent probes, such as Taqman probes 12 , are usually needed to determine the specificity of amplification. Therefore, isothermal amplification of RNA, such as nucleic acid sequence-based amplification [13][14][15] , rolling-cycle amplification 16,17 and loopmediated isothermal amplification 18,19 have emerged as alternative amplification techniques. As the above mentioned reactions can be preceded at a constant temperature, there is no need of specialized instruments for RNA detection, and in addition, they have potential for 'on-site' testing.…”

“…Two kinds of catalytic DNA sequences, RNA-cleaving DNAzyme (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) 26,27 and G-quadruplex horseradish peroxidase-mimicking DNAzyme (PW17) [28][29][30] were applied in our research. The 10-23 RNAcleaving DNAzyme has been reported to cleave any purinepyrimidine (RY) junction under simulated physiological conditions 26 ; hence, it was applied in our strategy for the cleaving of target RNA molecule to initiate the following exponential amplification.…”