title: Post-PCR multiplex fluorescent ligation detection assay and flow cytometry for rapid detection of gene-specific translocations in leukemia.
abstract: 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 a (PML-RARalpha) 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-RARalpha 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.
authors.affiliation: Department of Pathology, University of New Mexico, Health Science Center, Albuquerque, NM, USA.
authors.forename: I-Ming
authors.initials: IM
authors.lastname: Chen
authors.forename: Artemis
authors.initials: A
authors.lastname: Chakerian
authors.forename: David
authors.initials: D
authors.lastname: Combs
authors.forename: Kelly
authors.initials: K
authors.lastname: Garner
authors.forename: David S
authors.initials: DS
authors.lastname: Viswanatha
mesh_terms: D045744:Cell Line, Tumor; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D017403:In Situ Hybridization; D007938:Leukemia / Q000235:genetics*; D015345:Oligonucleotide Probes; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014178:Translocation, Genetic
chemical_list: D017931:DNA Primers; D004273:DNA, Neoplasm; D015345:Oligonucleotide Probes; D015514:Oncogene Proteins, Fusion