Bgl II‐based panhandle and reverse panhandle PCR approaches increase capability for cloning der(II) and der(other) genomic breakpoint junctions of MLL translocations

Panhandle PCR techniques to amplify known sequence flanked by unknown sequence have been useful for MLL genomic breakpoint junctions and fusion transcripts because MLL has a large number of partner genes. However, genomic panhandle PCR approaches are impeded when the restriction fragment that contains the breakpoint junction is too large to amplify. We devised new panhandle PCR approaches for MLL genomic breakpoint junctions that create the template from Bgl II restriction fragments by attaching MLL sequence to a Bgl II site in the partner gene. This leads to the annealing of MLL and its complement in the handle and creates an intrastrand loop containing the breakpoint junction sequence for amplification with primers all from MLL. Bgl II panhandle PCR for der(11) breakpoint junctions was accomplished by ligating a phosphorylated oligonucleotide containing a Bgl II overhang and sequence complementary to MLL exon 7 to the 3′ ends of Bgl II digested DNA, and forming the template from the sense strand of DNA. In Bgl II reverse panhandle PCR for der(other) breakpoint junctions, a phosphorylated oligonucleotide containing a Bgl II overhang and the complement of antisense sequence in MLL exon 10 was ligated to the 3′ ends of Bgl II digested DNA, and the template was formed from the antisense strand of DNA. These approaches amplified 5 ′‐ MLL‐MLLT4‐3 ′ and 5 ′‐ AFF1‐MLL‐3 ′ breakpoint junctions. The former is significant because few t(6;11) genomic breakpoint junctions have been sequenced. Bgl II panhandle PCR approaches increase the possibilities for cloning MLL genomic breakpoint junctions where there is heterogeneity in partner genes and breakpoint locations. © 2006 Wiley‐Liss, Inc.