RAPID SYNTHESIS OF HYBRID RNA MOLECULES ASSOCIATED WITH LEUKEMIA-SPECIFIC CHROMOSOMAL TRANSLOCATIONS

A number of gene arrangements have been described as characteristic ab normalities associated with different types of leukemia, and this list is still growing. In view of the biological, clinical and prognostic relevance of the pathological fusion products, techniques permitting t heir detection are of paramount importance in the clinical setting. In same instances, permanent leukemic cell lines carrying the abnormalit y of interest are available for the establishment and standardization of molecular assays. For a number of newly discovered gene rearrangeme nts, however, this may not be the case. It is therefore of great inter est for clinical laboratories to have alternative technical possibilit ies for the set-up of standardized molecular tests. This problem provi ded the stimulus to design a simple and rapid method for in vitro gene ration of chimeric RNA molecules corresponding to pathological fusion transcripts typical for chromosomal translocations in leukemias. Two s eparate fragments are generated in a four-primer multiplex PCR. Due to a PCR-generated overlap, a chimeric fragment can be synthesized in a second round of PCR. This PCR product is then purified with the help o f magnetic beads. Due to the SP6 promotor sequence incorporated during the second round of PCR, transcription into RNA is easily facilitated while the template DNA is still bound to the solid phase. Following t his strategy we were able to synthesize the fusion transcripts m-BCR/A BL, CBF beta/MYH11, and MLL/AFp1 which are the molecular equivalents o f t(9;22)(q34,q11), inv16(p13;q22) and t(1;11)(p32;q23), respectively. The chimeric RNA will be useful as a control template in diagnostic R T-PCR strategies. It can also be further processed in translation syst ems leading to the corresponding chimeric oncoprotein. This approach c an be easily used to create any hybrid RNA of interest.