An efficient and general method for sequencing immunoglobulin mRNAs.
By using two oligonucleotide primers, one complementary to a short DNA segment in the first constant region exon and second complementary to a short segment in the variable region, we have developed a highly efficient method for sequencing immunoglobulin mRNAs. Using the first primer full length immunoglobulin-specific cDNA was synthesized and separated on a denaturing acrylamide/urea gel. The desired band was isolated from the gel and sequenced by Maxam/Gilbert method. Using the sequence generated, a second oligonucleotide primer complementary to a short segment in the variable region was synthesized and similarly used to complete the remaining sequence of the variable region, the signal peptide and the 5'-untranslated region. We have used this method to determine the complete nucleotide sequence of heavy and light chain mRNAs from two anti-idiotypic hybridomas. The method is generally applicable to sequencing immunoglobulin mRNAs from both myelomas and hybridomas especially in instances involving the expression of more than one heavy or light chain mRNAs as well as instances involving additional transcripts from aberrantly rearranged immunoglobulin genes.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D006825:Hybridomas / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D007136:Immunoglobulins / Q000235:genetics*; D051379:Mice; D008805:Mice, Inbred A; D011061:Poly A / Q000235:genetics; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*
D007147:Immunoglobulin Light Chains; D007145:Immunoglobulin kappa-Chains; D007136:Immunoglobulins; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA