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, us ing 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 se gments of the V(lambda)1 and V(lambda)2 genes. Furthermore, an amplifi cation-and cloning-associated artifact exchanged sequences between mut ational variants of V(lambda)1 genes. These PCR artifacts interfere wi th the analysis of somatic hypermutation of Ig genes. An alternative m ethod that avoids these artifacts is suggested which involves the ampl ification of individual V-lambda genes from single cells.