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Post-PCR Multiplex Fluorescent Ligation Detection Assay

Post-PCR Multiplex Fluorescent Ligation Detection Assay
We describe a novel method to detect specific polymerase chain reaction (PCR) target amplicons, involving thermostable ligation of fluorescent and biotinylated oligonucleotides, microparticle bead capture of the ligated products, and flow cytometric analysis. This approach, termed fluorescent ligation detection reaction (f-LDR) is more rapid and cost-effective than oligoprobe Southern blot hybridization (SBH). A standard f-LDR protocol was developed to detect the leukemia-associated chimeric transcripts bcr-abl and promyelocytic leukemia-retinoic acid receptor α (PML-RARα) in 2 multiplex and multicolor assays. The f-LDR platform was 100% specific and demonstrated comparable or better sensitivity than standard oligoprobe SBH. The usefulness of f-LDR was evaluated in 94 posttherapy samples from 13 patients with acute promyelocytic leukemia with the PML-RARα gene fusion. The f-LDR method was highly concordant (93%) with oligoprobe SBH; essentially all discrepancies were noted to be due to the enhanced sensitivity of f-LDR. We conclude that f-LDR is a highly specific and sensitive post-PCR method with wide potential application.

Rapid and accurate identification of prognostically relevant gene fusions arising from chromosomal translocations is critical to establish a specific diagnosis in many pediatric and adult acute leukemias, as well as in chronic myeloid leukemia. These molecular markers are equally useful for guiding more tailored antileukemic therapy and further provide sensitive, disease-specific targets for posttherapeutic minimal residual disease monitoring. Although a variety of specialized methods commonly are used to detect these abnormalities, including cytogenetics and fluorescence in situ hybridization, reverse transcription-polymerase chain reaction (RT-PCR)-based techniques are used widely for their rapidity and the ability to evaluate minimal residual disease. Standard post-PCR analysis by agarose gel electrophoresis may suffice for initial identification of a chimeric gene fusion abnormality; however, many laboratories use additional mea-sures to confirm the specificity of the amplified PCR product, such as Southern blot transfer with oligonucleotide probe hybridization (SBH) for the sequence of interest.

Zhang et al described alternative oligonucleotide detection methods, based on simple solution phase binding of a fluorescent probe to a PCR target, and Gaffney et al described a novel application of thermostable oligonucleotide ligation termed fluorescent ligation detection reaction (f-LDR). The f-LDR approach was used successfully to distinguish the 2 major transcriptional products of the SYT-SSX chimeric gene fusion resulting from the t(X;18) abnormality in synovial sarcoma. The specificity of this approach was illustrated by the ability of closely related fluorescent ligation primers to accurately distinguish between 2 highly homologous target amplicon sequences, representing the SYT-SSX1 or SYT-SSX2 fusion genes. In the present effort, we sought to apply the f-LDR approach to more complex leukemia-associated gene fusions, reasoning that this technology would provide rapid and specific detection of these abnormalities following PCR amplification and the ability to potentially multiplex reagents for simultaneous identification of alternative fusion transcripts. This technique emphasizes high specificity, enhanced sensitivity, and the ability to process multiple samples from RNA isolation to final analysis in the same day. Furthermore, this method could be applicable to other molecular diagnostic targets in which rapid and specific detection of PCR products is required.

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