Types and major features of RCA reactions

In its original formulation,[1,2] the RCA reaction involves numerous rounds of isothermal enzymatic synthesis in which DNA polymerase extends a circle-hybridized primer by continuously progressing around the circular

DNA probe of several dozen nucleotides to replicate its sequence over and over again (Fig. 1A). This process is characterized by linear kinetics, easily yielding in one hour up to several thousands of sequence-complementary tandem repeats of an original DNA minicircle. These amplification products generally exhibit a wide, essentially continuous distribution over length and are normally seen in gel-electrophoretic images as a broad smear of high-molecular weight DNAs (Fig. 2, lane 2). The single-stranded nature of amplicons in case of linear RCA may be beneficial for subsequent manipulations with these DNAs towards their detection.[4]

A more complicated version, the double-primed RCA, called hyperbranched,[4] ramification[5] or cascade RCA,[6] operates with a pair of different primers. One primer is complementary, as in the linear RCA, to a DNA minicircle, whereas the other is targeted to the repeated, single-stranded DNA sequences of the primary RCA product.[4-6] Consequently, the double-primed RCA proceeds as a chain reaction with geometric kinetics featuring a ramifying cascade of multiple-hybridization, primer-extension, and strand-displacement events involving both primers (Fig. 1C). As a result, a discrete set of concatemeric double-stranded DNA (dsDNA) fragments is formed, yielding the distinct, ladder-type gel-electro-phoretic bands (Fig. 2, lane 3).

Sometimes, this reaction produces amplicons so long that they cannot move through the gel during common gel electrophoresis (see lane 4 in Fig. 2). In terms of the degree of amplification, the geometric RCA is more potent, as compared with its linear alternative, yielding 109 or more copies of a circular sequence in about an hour.[4,6] Accordingly, it enables a bioanalyst to readily detect just a few probe molecules.[4-8] The double-primed RCA is usually performed at elevated temperatures by thermostable DNA polymerases, although in some cases it is performed by common polymerase enzymes at physiological or ambient temperatures.

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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