Improved PCR method for detecting monoclonal immunoglobulin heavy chain rearrangement in B cell neoplasms.
OBJECTIVE
To develop a simple, optimised, polymerase chain reaction (PCR) based method for detecting the rearranged immunoglobulin heavy chain (IgH).


METHODS
Using as primers oligonucleotides (Fr2A, Fr2B) homologous to the conserved sequences to the framework II region and the joining (JH) region, 25 patients with B cell lymphoproliferative disorders, previously characterised by Southern blotting, and three patients with light chain myeloma were studied.


RESULTS
The PCR product from a polyclonal B cell population showed a broad band when analysed on a 3% agarose gel; DNA from B cell lines and B lymphoproliferative disorders showed a discrete band. Specificity of the amplification was confirmed by cloning and sequencing the amplified product as well as by Southern blotting with an internal probe homologous to the framework 3 region. Primers Fr2A and Fr2B detected monoclonality in three patients with light chain myeloma, while primers directed against the FrIII region showed a polyclonal response.


CONCLUSIONS
Deletions and extensive somatic mutations within the FrIII region may give false negative results with primers homologous to the region. A PCR using the method described, with a repertoire of primers homologous to the FrII and FrIII regions, will therefore increase the frequency of detection of monoclonality.
Ramasamy
I
I
Department of Biochemistry, Repatriation General Hospital, Daw Park, SA.
Brisco
M
M
Morley
A
A
D000368:Aged; D001402:B-Lymphocytes / Q000502:physiology; D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D008232:Lymphoproliferative Disorders / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D007143:Immunoglobulin Heavy Chains; D004247:DNA