Practical RCA formats

Generally, the RCA-based diagnostics can be classified into two groups: some of them operate with the preformed circular probes [Fig. 1D and E for the peptide nucleic acid (PNA)-assisted, nick-induced RCA and immuno-RCA,

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PNA openers

Fig. 1 Schematics of different RCA processes; the arrowhead symbolizes the DNA polymerase. Given a small, < 100-nt size of DNA minicircles used in RCA and the strong rigidity of dsDNA fragments with these lengths, only part of the circular probe can be base-paired at any given time. Consequently, the geometry of RCA-generating complexes resembles a fiddlestick. (A) The RCA reaction carrying on a free DNA minicircle with the use of a single primer. If the DNA target is used to prime the RCA reaction, amplification products are fixedly linked to target molecules (see schematics D, for example). For surface-attached targets, these products will be immobilized on the solid phase. (B) RCA-based diagnostics of probe amplification with an in situ circularized linear oligonucleotide probe and a target-unrelated primer (ligation-RCA/L-RCA). In some cases, the topological linkages between a DNA minicircle and the marker/target DNA site may affect the rolling replication. A circular probe should then be released from the DNA target following the hybridization. (C) Initial stages of the double-primed RCA.'4-6] In these reactions, the second primer, which is complementary to the original RCA product, is used. Here, the DNA polymerases capable of strand-displacement synthesis are necessary. (D) The RCA reaction, which proceeds on dsDNA if assisted by PNA openers and DNA nicking.'14-1 (E) In immuno-RCA, the 5' end of a primer is attached to a reporter antibody, which selectively binds to a test analyte immobilized on a solid surface.'7-

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PNA openers

Fig. 1 Schematics of different RCA processes; the arrowhead symbolizes the DNA polymerase. Given a small, < 100-nt size of DNA minicircles used in RCA and the strong rigidity of dsDNA fragments with these lengths, only part of the circular probe can be base-paired at any given time. Consequently, the geometry of RCA-generating complexes resembles a fiddlestick. (A) The RCA reaction carrying on a free DNA minicircle with the use of a single primer. If the DNA target is used to prime the RCA reaction, amplification products are fixedly linked to target molecules (see schematics D, for example). For surface-attached targets, these products will be immobilized on the solid phase. (B) RCA-based diagnostics of probe amplification with an in situ circularized linear oligonucleotide probe and a target-unrelated primer (ligation-RCA/L-RCA). In some cases, the topological linkages between a DNA minicircle and the marker/target DNA site may affect the rolling replication. A circular probe should then be released from the DNA target following the hybridization. (C) Initial stages of the double-primed RCA.'4-6] In these reactions, the second primer, which is complementary to the original RCA product, is used. Here, the DNA polymerases capable of strand-displacement synthesis are necessary. (D) The RCA reaction, which proceeds on dsDNA if assisted by PNA openers and DNA nicking.'14-1 (E) In immuno-RCA, the 5' end of a primer is attached to a reporter antibody, which selectively binds to a test analyte immobilized on a solid surface.'7-

as examples], whereas others involve the circularization of hybridized linear probes by ligation followed by RCA (L-RCA; Fig. 1B). In the latter case, the in situ assembled DNA minicircles, called padlocks and earrings,'9-13-provide the corresponding DNA/RNA diagnostic assay with a higher sequence specificity, which is warranted by a multiple (at least dual) probe-target recognition and is also owing to the fact that mismatches close to the ligation point severely interfere with the ligation process. Importantly, padlocks and earrings ensure a higher stability of hybridization complex because of additional topological stabilization through the probe-target concatenation.'13] All this improves the hybridization stringency, allows one to more efficiently distinguish single-base sequence variants, and results in a highly localized hybridization/ amplification signal retaining the positional information.

Further localization of the RCA-generated signal to essentially single visible points can be reached by condensation of amplication products after their hybridization to labeled oligonucleotides, known as RCA-CACHET.[4] In this way, the RCA amplicons are tagged with fluorescent labels at multiple sites in the tandem RCA-amplified DNA sequence. Thus ''decorated'' ampli-cons can be compacted into tiny objects by cross-linking with multivalent proteins, such as streptavidin and antibodies, which bind to amplicon-incorporated tags. If necessary, further increase of the RCA-generated signal to a superexponential level can be achieved by combining the RCA and PCR reactions.'5- Until recently, the RCA reactions have been run only on the single-stranded DNA and RNA targets, but, with the aid of PNA openers, these reactions can now be performed with dsDNA.[12,14] In immuno-RCA assays, the attachment of a reporter antibody to an RCA primer makes it possible to extend the RCA-based diagnostics on the non-nucleic-acid analytes, including proteins, which can be detected with superior sensitivities, compared to conventional enzyme immuno-assays in ELISA and microparticle formats.'7-

The RCA-based analyses can be executed both as homogeneous assays in solution and as heterogeneous ''on surface'' assays, including the microtiter plate and microarray approaches for high-throughput genomics and

Fig. 2 Typical patterns of the RCA products generated with linear or geometric kinetics and resolved by gel electrophoresis (SYBR green or ethidium bromide staining). Usually, linear amplification is characterized by smearlike amplicons corresponding to essentially continuous distribution of single-stranded RCA products over length (lane 2). On the contrary, ladder-like amplicons are normally observed after geometric amplification representing linear concatemeric double-stranded copies of a circular template (lane 3). In some cases, most of the RCA products are so large that they cannot enter the gel (lane 4). Lane 1 corresponds to the size marker.

Fig. 2 Typical patterns of the RCA products generated with linear or geometric kinetics and resolved by gel electrophoresis (SYBR green or ethidium bromide staining). Usually, linear amplification is characterized by smearlike amplicons corresponding to essentially continuous distribution of single-stranded RCA products over length (lane 2). On the contrary, ladder-like amplicons are normally observed after geometric amplification representing linear concatemeric double-stranded copies of a circular template (lane 3). In some cases, most of the RCA products are so large that they cannot enter the gel (lane 4). Lane 1 corresponds to the size marker.

proteomics studies.[15'16] The RCA amplicons can be detected in several ways, such as fluorescence,[7] radio-labeling,1-10-1 UV absorbance, and gel electrophoresis1-4,12-1 by using either direct incorporation of various labels into the RCA products[10] or label-decorated amplicons.[4,7] Since it is not possible to even briefly describe this whole diverse range of RCA testing formats, the interested reader is referred to recent reviews[17,18] and corresponding original papers.

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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