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Use of padlock probes and isothermal amplification for genetic detection


Genetic detection, particularly at the single molecule level, has emerged as a leading field in molecular diagnostics since the vast accumulation of genetic information due to DNA sequencing. Genetic detection is particularly important in cancer diagnostics and prognosis, since cancer cells exhibit unique genetic signatures. Padlock probes are particularly suitable for this purpose due to their intrinsic specificity mediated by precise base pairing of 3' and 5' ends for ligation to occur. Moreover, isothermal amplification of ligated padlock probes provides a rapid and sensitive assay for genetic detection. We have investigated two isothermal amplification assays, namely rolling circle amplification (RCA)-loop mediated isothermal amplification (LAMP) and ramification amplification (RAM) in single molecule detection. RCA-LAMP was discovered to successfully detect 10 molecules of ligated padlock probe, and proved a more rapid and sensitive assay than RAM. In addition, a breakpoint in the 20 kb deletion in the 9p21 chromosome of Detroit 562 pharyngeal cancer cells was detected by RCA- LAMP. Herein, we also discuss the limitations and shortcomings of each assay and the application of isothermal amplification of ligated padlock probes in chronic lymphocytic leukemia (CLL)

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