Application

The practical uses of RAM assay to detect target nucleic acids in clinical samples have been demonstrated in several studies (Fig. 2). Zhang et al. have applied the RAM assay to detect Chlamydia trachomatis, a leading cause of sexually transmitted disease (STD) in the United States, in cervical specimens collected in PreservCyt cytological solution.[10] They demonstrated the RAM assay can detect as few as 10 C. trachomatis elementary bodies in less than 2 hr, comparable with those of Amplicor PCR and ligase chain reaction (LCR), and they further tested 30 clinical specimens and detected all positive samples confirmed by PCR and LCR. The RAM assay can be an alternative to PCR and LCR to detect sexually transmitted agents because of its simplicity and isothermal amplification nature. Furthermore, it is possible to screen simultaneously cervical intraepithelial lesions and to detect STD agents in a single collection vial. Zhang et al. have also successfully applied the RAM assay for detection of Epstein-Barr virus in human lymphoma specimens.[2] They demonstrated the feasibility of RAM to detect a rare DNA target in clinical specimens and achieved an analytical sensitivity of as low as 10 molecules, comparable to that of PCR.

Fluorescent-based RAM assays have been developed to monitor and quantify the RAM products in a real-time fashion in a homogeneous reaction. Zhang et al. have demonstrated that RAM products can be monitored in the presence of SYBR green, and SYBR green can be incorporated into RAM reaction for the real-time detection of RAM products. The major limitations of intercalating dyes are that it can only detect one target per reaction, and that the signal can also be generated from nonspecific polymerization products. Fluorescent molecular beacons can also be used for real-time RAM assay.[8,11] Amplifluor is used as a primer and the reverse sequence displaces the stem structure, resulting in the release of fluorescence.

The RAM mechanism has been applied to amplify large, circular DNA such as the M13 phage (30,000 bp) or plasmid DNA from single colonies or plaques.[8] Dean et al.[12] showed that using random primers and 29 DNA polymerase, circular DNA templates can be amplified up to 10,000-fold in a few hours. This procedure removes the need for lengthy growth periods for phage and plasmid as well as traditional DNA isolation methods. The amplified products can be used directly for DNA sequencing, in vitro cloning, library construction, and other molecular biology applications.

The RAM mechanism can also be used to amplify linear DNA, such as genomic DNA. Dean et al.[12] recently demonstrated that whole genomic DNA can be amplified with 3' thiophosphate-modified random hex-amer and 29 DNA polymerase. About 20-30 mg DNA can be generated from as few as 10 copies of genomic DNA in about 6 hr of incubation at 30°C; the average product length was >10 kb. The products can be used directly for sequence analysis, SNP detection, comparative genome hybridization, or loss of heterozygosity analysis.

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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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