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Cloning and assembly of immunoglobulin variable regions from hybridomas is now a widely used technology to raise single-chain Fv (scFv) with predefined specificities. Many hybridomas are prepared with fusion partners derived from the mouse myeloma MOPC-21. MOPC-21 produces a normal kappa (κ) light chain and an aberrant one with a nonfunctional VJ recombination (MOPC abVκ, Accession No. M35669) (1).The P3-X63-Ag8.653 and Sp2/O fusion cell lines have been selected from MOPC-21 for their nonexpression of the normal κ chain (9). However, MOPC-21 abVκ mRNA is still present in these cells. Sets of oligonucleotides designed to amplify variable regions cannot discriminate between the functional gene derived from the fused B cell and the MOPC21 transcript. Many groups reported its preferential amplification by reverse transcription polymerase chain reaction (RT-PCR) when attempting to clone the light chain of hybridomas derived from B cells fused to one of these fusion partners (6,10,11,13,16). We recently tried to clone the variable regions of the rat antibody 505.1, directed against the Escherichia coli Glucosamine 6-P Synthase (4). The 505.1 hybridoma was obtained by fusion of rat splenocytes with Sp2/O cells. The 505.1 variable regions of heavy chains (VH) and light chains (VL) were first PCR-amplified as previously described (12). Fragments were subsequently assembled to generate the VH-(GGGGS)3-VL insert, which was then cloned into the pSW1 vector (18). All of the clones sequenced were found to contain the aberrant Vκ derived from MOPC-21. Several methods have been proposed to avoid the cloning of the MOPC abVκ; however, they are laborintensive or only moderately efficient (6,10,11,13,16). Here, we present an efficient and simple method to specifically inhibit the amplification of this aberrant gene with a peptide nucleic acid (PNA) matching its CDR3. PNAs are analogs of oligodeoxynucleotides (2,7,14) that form highly stable complexes with complementary DNA but cannot function as primers for DNA polymerases (15). We designed a PNA (H2N-CGTGTAAGCTCCCTA-H; PE Biosystems, Framingham, MA, USA [formerly PerSeptive Biosystems]) to specifically block the PCR elongation of the MOPC abVκ in conditions where the functional Vκ from the hybridoma would be amplified from cDNA. Criteria for designing the PNA were its high specificity based on the analysis of the Kabat database (8) and an elevated melting temperature (Tm) (66°C) determined as recommended by the manufacturer. Six nucleotides separate the carboxy-terminus of the PNA from the 3′ end of the antisense Vκ primer, a number that could be critical for PCR clamping (15) but that was nonetheless mandatory based on the two defined criteria.

biotechniques/biotechniques

Selective PCR Amplification of Functional Immunoglobulin Light Chain from Hybridoma Containing the Aberrant MOPC 21-Derived Vκ by PNA-Mediated PCR Clamping