Demonstration of array-based analysis for highly multiplexed PCR assays: Application to detection of IGH@-BCL2 translocations in FFPE follicular lymphoma specimens

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Abstract

We demonstrate an approach that allowed rapid development of a robust assay for the detection of chromosomal translocations. The method includes highly multiplexed PCR with analysis of the PCR products performed by array detection. As proof of principle, we applied this approach to the detection of IGH@-BCL2 translocations in DNA prepared from FFPE specimens. This translocation and specimen type were chosen because of the known difficulties associated with PCR-based detection of this lesion and the additional loss of sensitivity associated with FFPE samples. The multiplex PCR with array detection method detected the IGH@-BCL2 translocation in 26 of 36 FFPE follicular lymphoma specimens, whereas the BIOMED-2 assay detected 13 of 36 specimens. This increased sensitivity was the result of both the increased density of BCL2 primers and identification of PCR products by low-density array. The method was specific and allowed mapping of the BCL2 break point in all cases. The method detected the IGH@-BCL2 lesion when the tumor DNA was diluted more than 1:20 in normal DNA but not when it was diluted more than 1:100. This sensitivity allows detection of diagnostically relevant levels of IGH@-BCL2 but will not detect the rare cells with IGH@-BCL2 translocations in healthy individuals.

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Spence, J. M., Rothberg, P. G., Wang, N., & Burack, W. R. (2011). Demonstration of array-based analysis for highly multiplexed PCR assays: Application to detection of IGH@-BCL2 translocations in FFPE follicular lymphoma specimens. Journal of Molecular Diagnostics, 13(3), 252–262. https://doi.org/10.1016/j.jmoldx.2010.11.019

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