Detection of immunoglobulin heavy-chain gene rearrangements by Southern blot analysis: recommendations for optimal results.
Southern blot analysis of immunoglobulin (Ig) and T-cell receptor genes has proven to be important for detection of clonal rearrangements in patients with lymphoproliferative diseases. To improve the detection of clonal Ig heavy-chain (IgH) gene rearrangements, we carefully determined the precise restriction map of the joining (J)H and constant (C)mu region of the IgH locus, and evaluated relevant combinations of restriction enzymes with JH and C mu probes. Our extensive Southern blot analyses revealed that rearrangements in the JH region are optimally detectable by use of a JH probe in combination with at least two restriction enzyme digests which are not affected by polymorphisms, and which produce small germline bands (e.g. BglII and BamHI/HindIII), thereby reducing the chance of comigration of germline and/or rearranged bands. Application of a JH or a C mu probe in combination with BamHI or EcoRI digests should be avoided, because of the large size of the restriction fragments and the occurrence of polymorphisms. Comparison of different types of JH probes demonstrated that optimally reproducible signals, independent of the rearranged JH gene segment, are only obtained if the JH probe is complementary to the 3' flanking sequences of the JH gene region, such as our IGHJ6 probe.
Beishuizen
A
A
Department of Immunology, University Hospital Dijkzigt/Erasmus University, Rotterdam, The Netherlands.
Verhoeven
M A
MA
Mol
E J
EJ
Breit
T M
TM
Wolvers-Tettero
I L
IL
van Dongen
J J
JJ
D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods; D015342:DNA Probes; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D005803:Genes, Immunoglobulin; D006801:Humans; D015448:Leukemia, B-Cell / Q000175:diagnosis / Q000235:genetics; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D015183:Restriction Mapping
D015342:DNA Probes