Sequencing technology detection probes

Variations If the sequence difference between miR family members resides in the last 6 base pairs, utilization of fluorescent probes (such as Taqman technology, as shown in Chen et al., 2005) will give optimal specificity. In the example in Fig. 6.4A and B, both let-7c and let-7a are detected, but because both target the RAS family (Johnson et al, 2005), there was no need to distinguish between these family members in this experiment. 

In situ hybridization may be defined as the detection of nucleic acids in situ in cells, tissues, chromosomes, and isolated cell organelles. The technique was described in 1969 by two separate groups who demonstrated repetitive riboso-mal sequences in nuclei of Xenopus oocytes using radiolabeled probes (1,2). Refinements in recombinant DNA technology and the development of nonisotopic probe labeling and detection (3) obviate the need for radiation protection and disposal facilities, and have converted nonisotopic in situ hybridization (NISH) from a purely research technique to one that can be used in routine laboratory testing. 

The application of in situ hybridization (ISH) has advanced from short lived, non-specific isotopic methods, to very specific, long lived, multiple color fluorescent-ISH probe assays (FISH). Improvements in the optics, filter technology, microscopes, cameras, and data handling by software, have allowed for a cost effective FISH setup to be within reach of most researchers. The application of mFISH (multiplex-FISH), coupled to the advances in digital imaging microscopy, have vastly improved the capabilities for non-isotopic detection and analysis of multiple nucleic acid sequences in chromosomes and genes (1). 

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