Principle Of The Lamp Method Original LAMP Method

The LAMP method is performed with two inner primers, forward inner primer (FIP) and backward inner primer (BIP), and two outer primers, F3 and B3 primers. Forward inner primer has the same sequences as the F2 region and the F1C region of the target DNA, while BIP has the same sequences as the B2 region and the B1C region. The mechanism of the method and the expected reaction steps are illustrated in Fig. 2. The reaction involves three steps—''stating material producing step,'' ''cycling amplification step,'' and ''elongation step.''

First, for stating material producing step, the inner primer BIP hybridizes to B2c in the target DNA and initiates complementary strand synthesis (structure 1). The outer primer B3, which is a few bases shorter and is present at a low concentration, slowly hybridizes to B3c in the target DNA and initiates strand displacement DNA synthesis that releases a BIP-linked complementary strand (structure 2). Next, the inner primer FIP hybridizes to F2c in the released single-stranded DNA, and outer primer F3 hybridizes to F3c, releasing DNA as a dumbbell-like structure (structures 3 and 4).

The dumbbell-like DNA (structure 4) is quickly converted to stem-loop DNA (structure 5) by self-primed DNA synthesis. This stem-loop DNA then serves as the starting material for LAMP cycling, which is the second stage of the reaction (cycling amplification step; structures 4, 5, 6, and 7).

The DNA derived from structure 5 is also involved in ''elongation step'' of the reaction. Thus the 3' region of structure 5 is released by BIP-primed DNA synthesis,

Fig. 2 Schematic representation of the LAMP mechanism. In the first step, the starting structure is generated with dumbbell-like DNA (structure 4) being formed. Then, the starting structures are cyclically produced in cycling amplification step. The elongation reaction starts from the subproducts (structures 5 and 7) of the cycling amplification step, generating products of various sizes. F2c and B2c regions on the target gene are hybridized by the inner primers. Structure 11 is generated by the reaction starting from the loop primers. The region between F1 and F2 (or B1c and B2c) is hybridized by the loop primer. Dotted arrows represent continuous reactions. (View this art in color at www.dekker.com.)

Fig. 2 Schematic representation of the LAMP mechanism. In the first step, the starting structure is generated with dumbbell-like DNA (structure 4) being formed. Then, the starting structures are cyclically produced in cycling amplification step. The elongation reaction starts from the subproducts (structures 5 and 7) of the cycling amplification step, generating products of various sizes. F2c and B2c regions on the target gene are hybridized by the inner primers. Structure 11 is generated by the reaction starting from the loop primers. The region between F1 and F2 (or B1c and B2c) is hybridized by the loop primer. Dotted arrows represent continuous reactions. (View this art in color at www.dekker.com.)

and then self-primed DNA synthesis generates stem-loop DNA with a stem that is elongated to twice as long (structure 8). This reaction also occurs from structure 7 (following reactions not shown). Because the stem loop—including the single-stranded region containing the target sequence of the primer—is present in each structure, this reaction continues until the decay of the substrates or primers. The final products are a mixture of stem-loop DNAs of various stem lengths and cauliflower-like structures, with multiple loops induced by annealing between alternately inverted repeats of the target sequence on the same strand. When the products are electrophoresed on agarose gel, followed by staining with an intercalating dye such as EtBr, many bands of different sizes are detected (Fig. 1A).

Getting Started With Dumbbells

Getting Started With Dumbbells

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.

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