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Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family
Author(s) -
Chardès Thierry,
Villard Sylvie,
Ferrières Gaelle,
Piechaczyk Martine,
Cerutti Martine,
Devauchelle Gérard,
Pau Bernard
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)00649-3
Subject(s) - immunoglobulin light chain , paratope , oligonucleotide , microbiology and biotechnology , biology , gene , gene family , antibody , immunoglobulin heavy chain , dna sequencing , genetics , monoclonal antibody , primer (cosmetics) , chemistry , genome , organic chemistry
We have designed two original sets of oligonucleotide primers hybridizing the relatively conserved motifs within the immunoglobulin signal sequences of each of the 15 heavy chain and 18 kappa light chain gene families. Comparison of these 5′ primers with the immunoglobulin signal sequences referenced in the Kabat database suggests that these oligonucleotide primers should hybridize with 89.4% of the 428 mouse heavy chain signal sequences and with 91.8% of the 320 kappa light chain signal sequences with no mismatch. Following PCR amplification using the designed primers and direct sequencing of the amplified products, we obtained full‐length variable sequences belonging to major (V H 1, V H 2, V H 3, Vκ1 and Vκ21) but also small‐sized (V H 9, V H 14, Vκ2, Vκ9A/9B, Vκ12/13, Vκ23 and Vκ33/34) gene families, from nine murine monoclonal antibodies. This strategy could be a powerful tool for antibody sequence assessment whatever the V gene family before humanization of mouse monoclonal antibody or identification of paratope‐derived peptides.