Abstract:
A method based on differences in electrophoretic mobility of RNA transcripts made from polymerase chain reaction (PCR) products was used for differentiation among isolates of Prunus necrotic ringspot virus (PNRSV). A T7 RNA polymerase promoter was attached to amplified viral sequences by PCR. The PCR products than served as a template for transcription. Single-stranded (ss) transcripts originated from different PNRSV isolates, varied in their electrophoretic mobility in polyacrylamide gel, presumably because of transcript conformation polymorphism (TCP). Polymorphism of heterologous duplexes of RNA transcripts of PNRSV isolates was also demonstrated. Complementary RNA transcripts were copied from PCR products of PNRSV isolates to which the T7 RNA promoter was added on either the 3'- or 5'-ends. Heterologous duplexes made from complementary RNA transcripts originated from several virus isolates differed in their electrophoretic mobility. This polymorphism is hypothesized to be conferred by conformational variations among the transcript duplexes and was termed by us "doublestranded transcript conformation polymorphism" (ds-TCP). The resolution of ds-TCP is higher than ss-TCP and may serve as an additional tool in virus strain differentiation.
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