Mutation detection of immunoglobulin V-regions by DHPLC

One of the key features in the affinity maturation of antibodies is somatic hypermutation of the variable regions of immunoglobulin genes. The mutations that occur in immunoglobulin genes are detected by direct sequencing of cloned polymerase chain reaction (PCR) products. The frequencies of mutations in vivo are generally high enough to provide sufficient numbers of point mutations in order to generate large databases that can be analyzed in various ways. Recently, the mechanisms of variable (V)-region hypermutation have been studied in tissue culture systems and transgenic mice where mutation occurs at frequencies that are ∼10-fold lower than the estimated in vivo rate. Identifying mutations by brute force sequencing of PCR products in comparative studies is limiting when trying to determine if there are statistically significant differences. Here we describe a high throughput technique that can facilitate the identification of immunoglobulin V-regions that contain one or more mutations before sequencing. This technique, known as denaturing high-performance liquid chromatography (DHPLC), utilizes a standard HPLC apparatus with a column that binds double-stranded DNA (dsDNA). In this study, we have successfully detected ∼90% of previously sequenced mutated V-regions by DHPLC. Our results show that we were able to detect mutations throughout a 321-base pair (bp) region of the Ricin 45 immunoglobulin (Ig) V-region. Also, with the use of this assay, we have been able to detect mutations in multiple clones of different immunoglobulin genes.