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  • Title: Single-stranded RNA probes generated from PCR-derived DNA templates.
    Author: Bales KR, Hannon K, Smith CK, Santerre RF.
    Journal: Mol Cell Probes; 1993 Aug; 7(4):269-75. PubMed ID: 8232343.
    Abstract:
    The following report outlines the use of the polymerase chain reaction (PCR) in combination with in vitro transcription to generate single-stranded radiolabelled RNA probes useful for nuclease protection and in situ hybridization experiments. Specific DNA fragments with bacteriophage promoter (T3 and/or T7) sequences at the 5' or 3' end are generated by repeated rounds of amplification. Following purification, these PCR-generated DNA products are used as templates for in vitro transcription with the correct DNA-dependent RNA polymerase. The resultant radiolabelled, single-stranded RNA (ssRNA) can be used for in situ hybridization, Southern or Northern blot analysis, and ribonuclease protection experiments. Sub-cloning or hydrolysis of large fragments is not required. Probes can be made from virtually any sequence using a variety of template sources.
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