Overview Technical Description

Ramification amplification (RAM),[1,2] also termed hyperbranched rolling circle amplification1-3-1 or cascade rolling circle amplification,1-4-1 utilizes a closed C-probe or padlock probe and a DNA polymerase under isothermal conditions. The choice of DNA polymerase is based on the following requirements: a good strand displacement activity in order to separate downstream DNA from the template strand and a high processivity (i.e., incorporating nucleotides continuously on a given primer without dissociating from the template), allowing the polymerase to synthesize several thousand nucleotide-long ssDNA from the ligated C-probe. In this type of amplification, two primers, one complementary to the C-probe (forward) and the other identical in sequence to a second binding site in the C-probe (reverse), are added to the reaction. An initial rolling circle primer extension process is initiated and a single-stranded DNA (ssDNA) molecule is generated (rolling circle amplification). As the ssDNA molecule grows, multiple reverse primers are able to bind to the ssDNA and initiate a second ''round'' of primer extension templated by the initial ''rolling circle'' products. Once an upstream primer-extension strand ''bumps into'' a bound downstream primer, the polymer-ase displaces the downstream bound primer along with any extended sequence that is attached to it. The displaced dsDNAs serve as templates for further primer extension and amplification (Fig. 1). Like the constant unfurling of streamers, multiple primer extensions are simultaneously in progress, resulting in a large ramified complex. The reaction end-products are multimeric dsDNAs of various lengths, including smaller units such as monomers, dimers, trimers, and so on. Because the displaced DNAs are single-stranded, primers can bind to them at a constant temperature, eliminating the need for thermal cycling to generate ssDNA (as in the case of polymerase chain reaction, PCR).

The power of the exponential amplification of RAM can be expressed using the following formula: x(2U) where U is the number of repeats generated from the initial closed C-probe and where x is the number of the primer pair used.[1] For example, if one primer pair is used (one forward and one reverse), the formula is 2U, indicating that RAM is an exponential amplification. Compared to PCR that can be expressed as 2n, where N is the number of temperature cycles, RAM has an equivalent power of amplification. However, adding additional primer pairs can further enhance amplification. With three primer pairs (three forward and three reverse), the formula is 3(2U), indicating that RAM can result in a far greater magnitude of amplification.1-5-1 When using the RAM assay, the limiting factor is the length of the initial ssDNA generated from the closed C-probe; at least 20 repeats of ssDNA from the closed C-probe are required to achieve 1 million-fold amplification.

The two most important factors that affect amplification rate are stability of the DNA-polymerase complex and accessibility of the primer to its binding sites as soon as they are available. It is possible that formation of the large ramifying DNA-polymerase complex may interfere with the interaction of primers to their binding sites and disrupt the DNA-polymerase complex. Therefore the organic solvent dimethyl sulfoxide (DMSO, which stabilizes the DNA polymerase)1-6-1 and a protein co-factor T4 gene 32 protein (which stabilizes single-stranded regions of DNA)[7] can be added to enhance primer binding to the growing ssDNA and stabilize the DNA-polymerase complex. The addition of these agents

Fig. 1 Schematic representation of RAM. A forward primer (") bound to a C-probe is extended by a DNA polymerase (•), generating a long ssDNA. Multiple reverse primers (/) bind to the nascent ssDNA as their binding sites become available. Each bound reverse primer extends and displaces the downstream primers and their extended products. The forward primer binding sites of the displaced ssDNA are then available for the forward primers to bind and extend similarly, thus forming a large ramifying DNA complex (RAM).

Fig. 1 Schematic representation of RAM. A forward primer (") bound to a C-probe is extended by a DNA polymerase (•), generating a long ssDNA. Multiple reverse primers (/) bind to the nascent ssDNA as their binding sites become available. Each bound reverse primer extends and displaces the downstream primers and their extended products. The forward primer binding sites of the displaced ssDNA are then available for the forward primers to bind and extend similarly, thus forming a large ramifying DNA complex (RAM).

significantly stimulates DNA synthesis of many DNA polymerases such as 29 DNA polymerase[5] and Bst DNA polymerase.[2] Other components that can also stimulate DNA synthesis of DNA polymerases in RAM include Triton X-100 and 5% tetramethyl ammonium oxalate.[8] The RAM assay offers many advantages over other amplification techniques. Primers can readily bind to ssDNAs displaced by DNA polymerase, enabling the reaction to be carried out under isothermal conditions, obviating the need for a thermal cycler. A multiplex assay can be designed using generic primers to amplify different probes with equal efficiency, resulting in a better multiplex capability than conventional PCR.[1,9] The RAM assay offers a uniform platform for both RNA and DNA detection with the ability to ligate both ends of the padlock probe regardless of the nature of target (DNA or RNA) eliminating the need for reverse transcription for detecting RNA.[9] Finally, ligation requires that both probe termini perfectly match in order

Fig. 2 Ramification amplification assay procedure. A clinical sample is lysed in a lysis buffer in the presence of capture probe, C-probe, and paramagnetic bead, and target DNA together with the bound C-probe are captured on a paramagnetic bead, allowing extensive washing to remove unbound C-probe and other components in the sample. The C-probe aligned on the target is linked together by a DNA ligase. RAM amplification is then carried out by the addition of forward and reverse primers and DNA polymerase. (View this art in color at www.dekker.com.)

Fig. 2 Ramification amplification assay procedure. A clinical sample is lysed in a lysis buffer in the presence of capture probe, C-probe, and paramagnetic bead, and target DNA together with the bound C-probe are captured on a paramagnetic bead, allowing extensive washing to remove unbound C-probe and other components in the sample. The C-probe aligned on the target is linked together by a DNA ligase. RAM amplification is then carried out by the addition of forward and reverse primers and DNA polymerase. (View this art in color at www.dekker.com.)

for hybridization to occur, permitting the detection of a single nucleotide polymorphism.

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.

Get My Free Ebook


Post a comment