16] In vitro transcription of cRNA from PCR-generated DNA fragments

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This chapter presents a method that provides a simple approach for the synthesis of a DNA template, containing RNA polymerase promoter sites, by using the polymerase chain reaction (PCR). A general schemata for the synthesis of a template with promoters at each end is shown in the chapter. Several RNA polymerases will transcribe from these DNA fragments, yielding complementary RNA (cRNA) probes of high quality. It is, thus, possible to generate a cRNA probe for any gene for which the sequence is known. Transcription from a PCR-generated DNA fragment has several advantages. Primers can be designed from any genes for which the sequence is known, enabling an investigator to prepare any probe of interest. There is no need to clone the fragment; both T7 and T3 polymerases transcribe efficiently from the PCR product containing specific promoter sequences. The probes of any length can be designed by varying the primer choices. It has been found that cRNA probes between 200 and 500 nucleotides are optimal for in situ hybridization. Both sense and antisense cRNA probes can be generated from the same construct, containing different promoter sequence at each end, by varying the RNA polymerase used for transcription.