BglII-based panhandle and reverse panhandle PCR approaches increase capability for cloning der(II) and der(other) genomic breakpoint junctions ofMLLtranslocations

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Panhandle PCR techniques to amplify known sequence flanked by unknown sequence have been useful forMLLgenomic breakpoint junctions and fusion transcripts becauseMLLhas 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 forMLLgenomic breakpoint junctions that create the template fromBglII restriction fragments by attachingMLLsequence to aBglII site in the partner gene. This leads to the annealing ofMLLand its complement in the handle and creates an intrastrand loop containing the breakpoint junction sequence for amplification with primers all fromMLL. BglII panhandle PCR for der(11) breakpoint junctions was accomplished by ligating a phosphorylated oligonucleotide containing aBglII overhang and sequence complementary toMLLexon 7 to the 3′ ends ofBglII digested DNA, and forming the template from the sense strand of DNA. InBglII reverse panhandle PCR for der(other) breakpoint junctions, a phosphorylated oligonucleotide containing aBglII overhang and the complement of antisense sequence inMLLexon 10 was ligated to the 3′ ends ofBglII digested DNA, and the template was formed from the antisense strand of DNA. These approaches amplified5′-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.BglII panhandle PCR approaches increase the possibilities for cloningMLLgenomic breakpoint junctions where there is heterogeneity in partner genes and breakpoint locations. © 2006 Wiley-Liss, Inc.

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