Antibodies, A Laboratory Manual, Second Edition, Edited by Edward A. Greenfield

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Creation of Recombinant Antibodies Using Degenerate Oligonucleotides

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In this method, mRNA is obtained from B cells as a source of heavy- and light-chain sequences. Typically, this procedure uses an existing hybridoma or myeloma. Extreme care is required to obtain high-quality mRNA, because mRNA is readily degraded by ubiquitous RNases that are common laboratory contaminants. Our laboratory has had good experience using RNAzol reagent and the method available from Molecular Research Center, Inc. Additional methods for preparing intact RNA and avoiding contamination with RNases are available in Chapter 6 of Green and Sambrook (2012). cDNA is prepared using purified mRNA and either oligo(dT) or a C-region 3 primer. The cDNA negative strand is complementary to the coding sequence of the heavy or light chain. A large number of papers have described the design of degenerate oligonucleotides for amplifying either mouse (LeBoeuf et al. 1989; Orlandi et al. 1989; Coloma et al. 1991; Wang et al. 2000, 2006; Rohatgi et al. 2008; Lim et al. 2010) or human (Larrick et al. 1989; Marks et al. 1991; Lim et al. 2010) heavy- and light-chain genes. However, inasmuch as there are approximately 100 variable gene segments in mouse or human, there may be a substantial advantage to having some knowledge regarding the antibody's subfamily when using this strategy. We have made use of anti-Ig agarosebased immunoprecipitation followed by SDS-PAGE and LC-MS/MS to identify amino acid sequences for some hybridomas (JR Dasch, unpubl.). This approach can aid in the design of a variable region primer or in selection of the appropriate degenerate primer for a specific variable gene subfamily.

Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition

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