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

Abstract

Aims-To establish a robust method of extracting DNA from paraYn wax embedded bone marrow trephine (PBMT) biopsies for the amplification of relatively long polymerase chain reaction (PCR) products. Method-Xylene and ethanol were used to remove paraYn wax from eight formalin fixed, EDTA decalcified PBMT biopsies and DNA extraction was performed using a Qiagen QIAamp tissue kit. The DNA samples were amplified using nine diVerent PCR primers sets, including those used to detect chromosomal translocations 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 successfully to generate DNA fragments up to 643 bp in length. Chromosomal translocations and immunoglobulin gene rearrangements were detected by PCR in some of the samples. Sequencing of the t(11;14) PCR product demonstrated the presence of chimaeric sequences, which included both bcl-1 and immunoglobulin heavy chain (IgH) gene sequences, consistent with the presence of this translocation. Conclusions-This method enables PCR analyses of PBMT biopsies that were not previously possible, oVering the prospect of improved accuracy of diagnosis and the monitoring of patients with bone marrow disease.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of the BIOMED-2 Concerted Action BMH4-CT98-3936

“…Numerous protocols for the extraction of DNA from paraffin-embedded tissue for PCR analysis have been published. 181,182,[185][186][187] Many of these aim to reduce DNA degradation and the coextraction of PCR inhibitors, but many of these methods require prolonged extraction procedures and can be unsuitable for use in the routine diagnostic laboratory. 176,177,188,189 DNA sample concentration and integrity were estimated by spectrophotometry and by comparison of sample DNA with known standards on agarose-gel electrophoresis.…”

Development and Clinical Application of a Panfungal PCR Assay To Detect and Identify Fungal DNA in Tissue Specimens

“…The PCR was negative for 10 specimens from eight patients with histologically proven IFIs ( Table 2, patients 55 to 62). Since all of these specimens were PE, the quality of DNA is likely to have been compromised by the routine processes required for histological examination, in particular the duration of contact with formalin fixative prior to paraffin embedding (41,42). Additionally, insufficient amounts of DNA may have been present for detection by PCR, since at least three of the specimens had scant fungal elements upon histological examination (Table 2).…”

Epstein‐Barr virus latent membrane protein 1 induces the matrix metalloproteinase‐1 promoter via an Ets binding site formed by a single nucleotide polymorphism: Enhanced susceptibility to nasopharyngeal carcinoma

“…34 In brief, 10 Â 10 mm paraffin sections were incubated with 1 ml xylene at 558C for 15 min. After centrifugation, the supernatant fluid was discarded.…”