Chimeric molecules created by gene amplification interfere with the analysis of somatic hypermutation of murine immunoglobulin genes.
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.
Ford
J E
JE
Department of Pathology, Stanford University School of Medicine, CA 94305.
McHeyzer-Williams
M G
MG
Lieber
M R
MR
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
D004274:DNA, Recombinant; D007135:Immunoglobulin Variable Region; D007146:Immunoglobulin lambda-Chains