A major problem of the LCR reaction is joining of blunt end duplexes to each other and to themselves, all of which can serve as templates for further amplification, although no real target DNA is present in the original sample and this poses significant limitations on the sensitivity of LCR. To overcome this problem, several modifications have been developed, including a combined PCR/LDR method and Gap-LCR (G-LCR). In PCR/LDR, the amplification step for LDR is PCR utilizing outside primers and employing only one pair of adjacent probes to detect the proper sequence in the PCR produced. This procedure does not produce double-stranded oligonucleo-tides to blunt end ligate. A recent study showed that PCR/LDR was more accurate than FISH in the quantification of HER2/neu gene copy numbers. Gap-LCR is a version of LCR where the probes are not adjacent but are separated by one to three bases on both the sense and antisense strand. A nonstrand displacing thermostable DNA polymerase, supplied with the necessary dNTPs, adds the missing bases to the 3' internal end of one of the two probes, thus initiating a template-dependent extension. Once the gap between the two probes is filled, the probes are once again adjacent and can be ligated. The same process occurs on the opposite strand (Fig. 2). By this modification, G-LCR prevents target-independent ligation very efficiently[20,21] and has the advantage of having the additive effect of polymerase and ligase on specificity. Excellent discrimination of mutations was achieved using multiplex G-LCR,[13,14] and a real-time G-LCR for rapid detection of p53 mutations in clinical specimens was recently described. Gap-LCR assays have been developed for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycobacterium tuberculosis (Abbott, IL, USA). Several studies have evaluated the usefulness of commercially available nucleic acid amplification assays, comparing G-LCR with PCR and SDA for detection of C. trachomatis and N. gonorrhoeae. In these comparative studies G-LCR proved to be a robust, specific, and sensitive technique.[24-29]
Was this article helpful?
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.