Rescue Strategy for Nonviable Hybridomas: Amplifying Heavy- and Light-Chain Sequences from Genomic DNA by Polymerase Chain Reaction

• Published in: Cold Spring Harbor protocols Vol. 2017; no. 10; p. pdb.prot093880
• Main Authors: Dasch, James R, Dasch, Amy L
• Format: Journal Article
• Language: English
• Published: United States 03.10.2017
• Subjects: DNA - genetics; DNA - isolation & purification; Hybridomas; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Light Chains - genetics; Molecular Biology - methods; Polymerase Chain Reaction - methods
• ISSN: 1559-6095
• DOI: 10.1101/pdb.prot093880

Many hybridoma researchers have lost an important hybridoma because of instability, mechanical breakdown of a freezer, or contamination. In many cases, however, it is possible to retrieve the heavy- and light-chain sequences from those hybridomas. Although most cloning methods associated with isolation of VH or VL gene sequences require the isolation of intact, nondegraded mRNA, alternative approaches based on genomic DNA have been reported. With genomic DNA, the DNA sample can remain relatively intact in cases in which the mRNA has been degraded. If intact DNA can be obtained from a nonviable hybridoma, the method described here can be used to recover the heavy- and light-chain sequences. The method uses PCR based on a combination of primers in the 5' untranslated region of VH and VL and in the JH and JL segments. Although a relatively large number of primers are required for this method, the ability to obtain a full-length V gene without having intact mRNA could be of general interest for rescuing lost hybridomas.