Isolation and characterisation of recombination events involving immunoglobulin heavy chain switch regions in multiple myeloma using long distance vectorette PCR (LDV-PCR).
Immunoglobulin class switching occurs as a result of recombination between pairs of switch region sequences located 5' to each constant heavy chain gene except Cdelta. In the B cell neoplasm multiple myeloma, tumour cells have generally undergone class switching and often contain oncogenic sequences translocated into switch regions, presumably as a result of aberrant switch recombination. We have developed a method (LDV-PCR) which combines long distance PCR with one-sided vectorette PCR that is capable of detecting and isolating both normal and aberrant switch recombination breakpoints from multiple myeloma cell lines and primary multiple myeloma tumour material. Using LDV-PCR we have directly cloned the translocation breakpoints present in two multiple myeloma cell lines and isolated a normal productive switch recombination event from a primary tumour. Furthermore, we have isolated a novel translocation t(14;22)(q32;q12) from a primary tumour sample and have demonstrated that internal deletions within switch regions can occur in multiple myeloma cells. Compared to a Southern blotting approach, LDV-PCR is simpler and more rapid to perform, allows the simultaneous detection and isolation of recombination events, and can also be applied to amounts of DNA which are too low to permit the conventional cloning of recombination breakpoints.
Proffitt
J
J
Institute of Pathology, University of Leeds, UK.
Fenton
J
J
Pratt
G
G
Yates
Z
Z
Morgan
G
G
D001483:Base Sequence; D002880:Chromosomes, Human, Pair 11; D002883:Chromosomes, Human, Pair 14; D002885:Chromosomes, Human, Pair 16; D002892:Chromosomes, Human, Pair 22; D005822:Genetic Vectors; D006801:Humans; D007134:Immunoglobulin Switch Region / Q000235:genetics*; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic; D014178:Translocation, Genetic; D014407:Tumor Cells, Cultured