title: Chimeric molecules created by gene amplification interfere with the analysis of somatic hypermutation of murine immunoglobulin genes.
abstract: We used the polymerase chain reaction (PCR) to amplify genes encoding murine immunoglobulin (Ig) lambda light-chain variable (V) regions, using DNA isolated from populations of germinal center B-cells, to study somatic hypermutation at this locus. Sequence analysis revealed that 30% of the amplified products were chimeric molecules consisting of segments of the V lambda 1 and V lambda 2 genes. Furthermore, an amplification- and cloning-associated artifact exchanged sequences between mutational variants of V lambda 1 genes. These PCR artifacts interfere with the analysis of somatic hypermutation of Ig genes. An alternative method that avoids these artifacts is suggested which involves the amplification of individual V lambda genes from single cells.
authors.affiliation: Department of Pathology, Stanford University School of Medicine, CA 94305.
authors.forename: J E
authors.initials: JE
authors.lastname: Ford
authors.forename: M G
authors.initials: MG
authors.lastname: McHeyzer-Williams
authors.forename: M R
authors.initials: MR
authors.lastname: Lieber
mesh_terms: D000818:Animals; D016477:Artifacts; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004274:DNA, Recombinant / Q000737:chemistry*; D005803:Genes, Immunoglobulin / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007146:Immunoglobulin lambda-Chains / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction
chemical_list: D004274:DNA, Recombinant; D007135:Immunoglobulin Variable Region; D007146:Immunoglobulin lambda-Chains