Ramification amplification (RAM) (Zhang et al., 1998, 2001), also referred to as hyperbranched rolling circle amplification (Lizardi et al., 1998) or cascade rolling circle amplification (Thomas et al., 1999), is a novel, isothermal DNA amplification that amplifies a C-probe exponentially through the mechanism of primer extension, strand displacement, and ramification. In contrast with RCA, the RAM assay uses 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). As with RCA, the initial rolling circle primer extension process generates a long ssDNA. However, as the ssDNA molecule expands, multiple reverse primers are able to bind to the growing ssDNA and initiate a second "round" of primer extension templated by the initial "rolling circle" products. Once a downstream primer encounters a bound upstream primer, the polymerase displaces the upstream bound primer along with any extended sequence that may be attached to it. The displaced ssDNAs serve as templates for further primer extension and amplification (Fig. 13.1C). Like the constant unfurling of streamers, multiple primer extensions take place simultaneously, resulting in a large ramified complex. Because the displaced DNAs are single-stranded, the binding of primers occurs at a constant temperature, thus obviating the need for thermocycling to generate single-stranded DNA, as in the case for LCR primers. Some of the properties of RAM are summarized in Table 13.1.
The practical use of RAM has been shown in several studies for detecting target nucleic acids in clinical samples. Zhang et al. (2002) were able to detect Chlamydia trachomatis in cervical specimens collected in PreservCyt cytological solution. Thirty clinical specimens were tested using the RAM assay, and the assay conferred accurate detection of all the positive samples that were confirmed by PCR and LCx. The RAM assay can detect as few as 10 C. trachomatis elementary bodies in less than 2 hrs. similar to the lower limit of detection for Amplicor PCR and LCx. Therefore, the RAM assay can serve as a feasible alternative to PCR and LCx for the detection of sexually transmitted infectious agents owing to its simplicity and isothermal amplification conditions.
The RAM assay was also used in the identification of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in food and human samples.
Combining magnetic bead-based DNA isolation, amplification of a stx2-specific C-probe by RAM and real-time fluorescence detection, Li and colleagues (Li et al., 2005) accurately identified all 27 pathogenic E. coli isolates producing Shiga toxin 2 from food and human samples, as previously confirmed by PCR using primers specific for the stx2 gene. One Shigella dysenteriae and three nonpathogenic E. coli were found negative by RAM assay. With respect to such application, the RAM assay provides a simple yet sensitive method that can be readily employed in clinical laboratories for the detection of food-borne pathogens and in meat product inspections.
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