Rapid amplification of cDNA ends (RACE) improves the PCR-based isolation of immunoglobulin variable region genes from murine and human lymphoma cells and cell lines.
The isolation of rearranged immunoglobulin (Ig) variable region (V) genes is usually performed by PCR with consensus primers binding to conserved regions within the V sequences. However, the isolation of Ig genes by this method is hampered in 15-35% by technical difficulties, mostly mismatches of oligonucleotide primers to V sequences. In order to obtain DNA sequences from V heavy chain (VH) genes which could not be amplified with consensus primers, we used a modified PCR technique, the rapid amplification of cDNA ends (RACE) PCR in combination with new heavy chain constant region primers for the isolation of human and murine VH genes. In comparison, consensus primer PCR with different sets of previously published oligonucleotide primers was used. Both methods were applied to isolate VH genes from murine B cell lymphoma (A20 and BCL1), myeloma (NS1) and hybridoma (SP6) cell lines and from freshly isolated human chronic lymphocytic leukemia and lymphoma cells. RACE PCR allowed the amplification and subsequent cloning of the complete VH gene in all cases. In contrast, consensus primer PCR failed to isolate the VH sequence of the murine A20 cell line; this was explained by a mismatch of consensus primers with VH sequences. When both PCR methods amplified VH sequences, the DNA sequences obtained were identical. Taken together, RACE PCR represents a reliable and versatile method for the isolation of VH genes from human and murine lymphoma cells, in particular if consensus primer PCR fails.
Doenecke
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Genzentrum, Universität München, Germany.
Winnacker
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EL
Hallek
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M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004273:DNA, Neoplasm / Q000302:isolation & purification*; D015321:Gene Rearrangement; D005803:Genes, Immunoglobulin; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D016393:Lymphoma, B-Cell / Q000235:genetics*; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D007135:Immunoglobulin Variable Region