Detection of four different 11q23 chromosomal abnormalities by multiplex-PCR and fluorescence-based automatic...

A nested polymerase chain reaction (PCR) protocol was developed for rapid detection of four different 11q23 abnormalities by a single PCR assay. During each of the two PCR rounds a sense primer located within exon 5 of the MLL gene at 11q23 was combined with four different antisense primers, each located within possible translocation partner genes at chromosomes 4, 6, 9, and 19, respectively. Except for the MLL primer all primers used during the second round of nested-PCR carried a characteristic fluorescence label at their 5'-end. Agarose gel analysis of the PCR products was sufficient to discriminate between the absence of any of the four MLL rearrangements and the presence of at least one of them. Discrimination of the four different MLL translocation partner genes was not possible by agarose gel analysis due to a molecular heterogeneity of the 11q23 breakpoints resulting in PCR products of variable size. For this reason, automatic fluorescence-based DNA-fragment analysis was used to exactly define the MLL translocation partner genes if a positive result had been obtained by agarose gel analysis. In patients with leukemia, this assay may enable a fast and highly sensitive detection of different 11q23 abnormalities, which usually correlate with poor clinical prognosis.