Abstract
Loop mediated isothermal amplification (LAMP) is one of the most popular isothermal DNA amplification techniques for research and commercial applications, enabling amplification of both DNA and RNA (with the assistance of reverse transcriptase). The LAMP mechanism is powered by strategic primer design and a strand displacement polymerase, generating products that fold over, creating loops. LAMP leads to generation of products of increasing length over time. These products containing multiple loops are conventionally called cauliflower structures. Existing literature on LAMP provides extremely limited understanding of progression of cascades of reactions involved in the reaction and it is believed that cauliflower structures of increasing length constitute a majority of the product formed in LAMP. This study presents a first of its kind stoichiometric and pseudo kinetic model to comprehend LAMP reactions in deeper depth by (i) classifying LAMP reaction products into uniquely identifiable categories, (ii) generating a condensed reaction network to depict millions of interconnected reactions occurring during LAMP, and (iii) elucidating the pathways for amplicon generation. Despite the inherent limitations of conventional stoichiometric modelling for polymerization type reactions (the network rapidly becomes too large and intractable), our model provides new theoretical understanding of the LAMP reaction pathway. The model shows that while longer length products are formed, it is the smaller length recycle amplicons that contribute more towards the exponential increase in the amount of double stranded DNA. Prediction of concentration of different types of LAMP amplicons will also contribute substantially towards informing design of probe-based assays.
Highlights
Loop-mediated isothermal amplification (LAMP) has been identified as a powerful isothermal nucleic acid amplification (NAA) technique over the past few decades
We present the first attempt at developing a stoichiometric and pseudo kinetic (SPK) model to provide deeper insights into the cascades of reactions occurring in LAMP with four primers (FIP, backward inner primer (BIP), F3 and B3)
Primer-based extension involves the annealing of inner primers (FIP or BIP) to their complementary region on the reactant DNA molecule, followed by extension by DNA polymerase
Summary
Loop-mediated isothermal amplification (LAMP) has been identified as a powerful isothermal nucleic acid amplification (NAA) technique over the past few decades. A literature search conducted on Web of Science revealed that over the last 5 years, LAMP has been the most frequently used technique for isothermal NAA with ~4x more publications compared to the second most popular technique (ESI Fig. S1). It has emerged as a popular technique to enable PCR-free molecular diagnosis of COVID19[6,7]. LAMP, as invented in 2000 by Notomi et al.[8], included four primers – a pair of inner primers (forward inner primer, FIP, and backward inner primer, BIP) and a pair of outer primers (forward outer primer, F3, and backward outer primer, B3). In general the reaction operates at a temperature in the range of 60-
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