PROTOTYPE RT AND PCR PROTOCOLS WITHOUT MAGNESIUM CHLORIDE USING NEOWATER AND DETECTION OF CANCER MEMBRANE ANTIGEN EP-CAM AND IDENTIFICATION OF CIRCULATING MICROMETASTATIC CANCER CELLS IN PERIPHERAL BLOOD (AND TISSUES) IN PATIENTS WITH CANCER OF EPITHELIAL ORIGIN

摘要

he invention concerns new prototype protocols for the reverse transcription (RT) and the reaction of the polymerase chain reaction (PCR) for the detection of the molecular marker Ep-CAM and the identification of circulatin micrometastatic cancer cells in peripheral blood of patients (and in tissues) suffering from cancer of epithelial origin. For the detection of circulatin micrometastatic cancer cells in the perihperal blood of patients (and in tissues) with cancer of epithelial origin is carried out with reaction PCR and analysis or the PCR products either through gel agarose electrophoresis or with hybridization in liquid or in membrane with any method, where is used, Neowater, specific primers, where the forward primer can be labeled with biotin in one of the ends and with specific internal probe for the hybridization. For the detection using hybridization in liquid or on membrane the labeling of the forward primer of the labeling of the internal probe could be varied. The newinvention decreases framatically the time and the quantity of the reagents, that are sufficient to carry our a tetection or test with the use of PCR or/and RT, while simultaneously is not used for the RT and PCR reaction any buffer, DTT for the RT and MgCl2 for the PCR and constitutes in the use of total RNA, that could be isolated with any common procedure. A part of the isolated total RNA will be used for the production of complementary DNA (cDNA) using Neowater, a commercial available reagent. A part of the produced cDNA will be used in the procedure of PCR, with the use of specific primers, Neowater, prototype thermical protocols and specific internal probe for the hybridization. Thus is detected the Ep-CAM and is carried out the identification of circulating micrometastatic cancer cells in peripheral blood or/and in tissues as well as in every other body fluid of patients with cancer of epithelial origin.