Molecular identification of the t(5;17)(q33;p13) fusion gene using 5′RACE-PCR.

(A) A schematic showing the 5′RACE method used to identify the breakpoint. Patient total RNA and mRNA and normal marrow stroma total RNA was reverse transcribed. A 5′ poly-A tail was appended on the resulting cDNA. Using a nested PCR the PDGFβR native or fusion gene was amplified using reverse PDGFβR primers (PDGFβR-Po [outer]/PDGFβR-Pi [inner]) and forward RACE primers (tailed poly-T Qo-T₍₁₇₎N and Qi [inner]; see “Patient and methods”). (B) After electrophoretic separation on 1% agarose gel a distinct band was seen in the nested PCR product from normal bone marrow stroma (arrow), whereas a smear was seen in PCR products derived from either patient total RNA or mRNA (box). TA cloning and sequencing of the bone marrow–derived band (arrow) revealed the native PDGFβR. The PCR smear (box) was TA cloned. (C) After screening and sequencing 24 TA-cloned colonies derived from the patients PCR product, a blast search in GenBank revealed a 454-nucleotide insert in a single colony with an in-frame fusion between rabaptin-5 and thePDGFβR. Shown is part of the sequence over the breakpoint (rabaptin-5 sequence in bold).