To detect a single nucleotide difference by LAMP-based SNP typing, both FIP and BIP were designed to contain SNP nucleotides on the target sequence at each 5' end. Figure 3A shows the basic principles of the LAMP cycling reaction with the WT primer set, using WT DNA (Fig. 3A, upper) and MUT DNA (Fig. 3A, lower) as templates.
When LAMP reaction using the WT primer set is carried out with WT DNA as the template, a dumbbelllike structure is generated with both ends being overlapped by self-annealing (Fig. 3A, upper). This dumbbelllike structure served as the starting point for cycling amplification step (Fig. 2, structure 4 or 6). DNA synthesis is initiated from the 3' end, using the structure itself as the template (similar to the original LAMP method). As a result, DNA strands consisting of inverted repeats of the target sequence are formed. When MUT DNA is used as the template, the 3' end of the dumbbelllike structure cannot self-anneal completely at its end (Fig. 3A, lower). Thus DNA synthesis is only primed from the 3' end of FIP or BIP that anneals to the loop region of the structure, resulting in the formation of double-stranded DNA and the halt of the LAMP cycling process. When DNA synthesis proceeded as a result of a miscopy and the dumbbell-like structure was produced, further DNA synthesis from the turnover structure is halted through the steps mentioned above. Because two primers are designed to recognize a single nucleotide difference of the dumbbell-like structure or its turnover structure, LAMP-based SNP typing could precisely discriminate single nucleotide differences.
To demonstrate the usefulness of LAMP-based SNP typing, we assessed the typing of CYP2C19, a gene belonging to the cytochrome P450 family and showing various polymorphisms. Although homology between the CYP2C subfamilies is very high, the LAMP method was capable of specifically amplifying CYP2C19 and detecting a single nucleotide difference during singlestep amplification, because the method uses four primers that recognize six regions and detects a single-base difference after each cycle of amplification. To demonstrate the value of LAMP, CYP2C19*1 (WT), 2C19*3 (G636A), 2C9, 2C18, and 2C8 genes were used as templates and the reaction was carried out in the presence of an intercalater for real-time detection (Fig. 3B). Only 2C19*1 alone was amplified when the 2C19*1 primer was used, while the 2C19*3 gene with a single nucleotide difference and genes of other 2C subfamilies were not amplified (Fig. 3B, left). Similarly, only the 2C19*3 gene was amplified and genes of the other 2C subfamilies were not amplified when the
2C19*3 primer was used (Fig. 3B, right). These results indicate that the LAMP reaction can detect a single nucleotide difference on a target gene when LAMP-based SNP typing is performed with human genomic DNA in the presence of many homologous family genes.
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The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.