A reliable approach for sequencing clone-specific CDRIII regions in B-cell lymphoma.
We report a reliable approach for sequencing lymphoma-specific CDRIII regions. CDRIII regions present in DNA prepared from routinely fixed and paraffin-embedded diagnostic lymph node material were amplified by the use of consensus VH and JH primers via PCR. PCR products were subcloned directly, without purification or modification of PCR fragments. Only small amounts of miniprep plasmid DNA of recombinant clones were required for cycle sequencing, resulting in autoradiograms of high quality. The easy and reproducible method which we describe has enabled us to determine the lymphoma-specific CDRIII region in 7/11 high-grade non-Hodgkin's lymphomas as well as in 3/3 cases of ALL and 1/1 case of a centroblastic/centrocytic lymphoma. The obtained sequence data can serve to generate lymphoma-specific oligonucleotides, which then can be used as PCR primers or hybridization probes for the detection of minimal residual disease in individual patients.
Straka
C
C
Department of Experimental Haematology, C.R.C. Paterson Institute for Cancer Research, Manchester, UK.
Pettengell
R
R
Pielmeier
A
A
Cross
M
M
Emmerich
B
B
Crowther
D
D
Testa
N G
NG
Dexter
T M
TM
D001483:Base Sequence; D003001:Cloning, Molecular; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D016393:Lymphoma, B-Cell / Q000235:genetics*; D008969:Molecular Sequence Data; D015203:Reproducibility of Results
D007135:Immunoglobulin Variable Region