Amplification of PCR products in excess of 600 base pairs using DNA extracted from decalcified, paraffin wax embedded bone marrow trephine biopsies

Aims-To establish a robust method of extracting DNA from paraffin wax embed ded bone marrow trephine (PBMT) biopsies for the amplification of relativel y long polymerase chain reaction (PCR) products. Method-Xylene and ethanol were used to remove paraffin wax from eight forma lin fixed, EDTA decalcified PBMT biopsies and DNA extraction was performed using a Qiagen QIAamp tissue kit. The DNA samples were amplified using nine different PCR primers sets, including those used to detect chromosomal tra nslocations t(11;14) and t(14;18), and clonal B cell populations. A t(11;14 ) PCR product of approximately 600 base pairs (bp) was sequenced using dye terminator cycle sequencing. Results-All eight DNA samples extracted from PBMT biopsies were amplified s uccessfully to generate DNA fragments up to 643 bp in length. Chromosomal t ranslocations and immunoglobulin gene rearrangements were detected by PCR i n some of the samples. Sequencing of the t(11;14) PCR product demonstrated the presence of chimaeric sequences, which included both bcl-1 and immunogl obulin heavy chain (IgH) gene sequences, consistent with the presence of th is translocation. Conclusions This method enables FCR analyses of PBMT biopsies that were not previously possible, offering the prospect of improved accuracy of diagnos is and the monitoring of patients with bone marrow disease.