Panhandle PCR for cDNA: A rapid method for isolation of MLL fusion transcripts involving unknown partner genes

Identifying translocations of the MLL gene at chromosome band 11q23 is impo rtant for the characterization and treatment of leukemia. However, cytogene tic analysis does not always find the translocations and the many partner g enes of MLL make molecular detection difficult. We developed cDNA panhandle PCR to identify der(ll) transcripts regardless of the partner gene. By rev erse transcribing first-strand cDNAs with oligonucleotides containing codin g sequence from the 5' MLL breakpoint cluster region at the 5' ends and ran dom hexamers at the 3' ends, known MLL sequence was attached to the unknown partner sequence. This enabled the formation of stem-loop templates with t he fusion point of the chimeric transcript in the loop and the use of MLL p rimers in two-sided PCR. The assay was validated by detection of the known fusion transcript and the transcript from the normal MLL allele in the cell line MV4-11. cDNA panhandle PCR then was used to identify the fusion trans cripts in two cases of treatment-related acute myeloid leukemia where the k aryotypes were normal and the partner genes unknown, cDNA panhandle PCR rev ealed a fusion of MLL with AF-10 in one case and a fusion of MLL with ELL i n the other. Alternatively spliced transcripts and exon scrambling were det ectable by the method, Leukemias with normal karyotypes may contain cryptic translocations of MLL with a variety of partner genes, cDNA panhandle PCR is useful for identifying MLL translocations and determining unknown partne r sequences in the fusion transcripts.