Rapid synthesis of hybrid RNA molecules associated with leukemia-specific chromosomal translocations.

A number of gene arrangements have been described as characteristic abnormalities 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 their detection are of paramount importance in the clinical setting. In some instances, permanent leukemic cell lines carrying the abnormality of interest are available for the establishment and standardization of molecular assays. For a number of newly discovered gene rearrangements, however, this may not be the case. It is therefore of great interest for clinical laboratories to have alternative technical possibilities for the set-up of standardized molecular tests. This problem provided the stimulus to design a simple and rapid method for in vitro generation of chimeric RNA molecules corresponding to pathological fusion transcripts typical for chromosomal translocations in leukemias. Two separate 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 of 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 this strategy we were able to synthesize the fusion transcripts m-BCR/ABL, CBF beta/MYH11, and MLL/AFp1 which are the molecular equivalents of 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 RT-PCR strategies. It can also be further processed in translation systems leading to the corresponding chimeric oncoprotein. This approach can be easily used to create any hybrid RNA of interest.