Cloning of t(2;14)(p13;q32.3) breakpoint by 5′ Sμ LDI-PCR.

(A) Ideogram showing strategy used for LDI-PCR cloning of 5′ Sμ breakpoints. Top panel represents normal chromosome 14 with class-switched IGH VDJ fragment; middle panel, der(2)t(2;14)(p13;q32.3); and lower panel, der(14)t(2;14)(p13;q32.3). Class-switching results in 5′ Sμ sequences being retained whereas 3′ Sμ sequences are deleted. Most IGHS translocation breakpoints occur within switch regions that have already undergone class-switching, leaving the 5′ Sμ intact on the other derivative chromosome. These sequences can then be used to amplify the translocated allele using 5′ Sμ primers. Black horizontal lines represent chromosome 14; red lines, chromosome 2. The vertical red arrow represents the translocation breakpoint. Vertical lines representHindIII sites. (B) The left panel shows 1.6 kb LDI-PCR product obtained with 5′ Sμ primers; lane M denotes molecular weight markers. The right panel shows southern blot of DNA from patient 4 and DNA from a healthy individual (lane C) digested withHindIII and probed with a 5′ Sμ probe.34 An arrow points to a 2.1 kb rearranged band in patient 4. This was shown to be an illegitimate IGHS recombination event by reprobing with a 3′ Sμ probe (data not shown). Given the location of the DNA primers within 5′ Sμ this is the size of the LDI-PCR product anticipated from the southern blot results. (C) Location of translocation breakpoints within 2p13. Breakpoints derived from either bacteriophage cloning or LDI-PCR are shown in red. All 3 cloned breakpoints fell within 125 bp of each other.