Branched DNA (bDNA) Technology for Direct Quantification of Nucleic Acids: Research and Clinical Applications

Abstract

Over the past decade a number of new technologies, including the polymerase chain reaction (PCR) (Mullis and Faloona, 1987), have emerged for the detection and quantification of nucleic acid molecules. Distinct among these is the branched DNA (bDNA) assay which, unlike its target amplification counterparts, uses a signal amplification scheme to enhance detection of physiologic concentrations of target nucleic acids. The basis of the bDNA assay involves the specific hybridization of bDNA and enzymelabeled oligonucleotide probes to target nucleic acids; this is described in detail in the accompanying chapter in this book, entitled “Branched DNA Technology for Direct Quantification of Nucleic Acids: Design and Performance.” The bDNA assay is inherently quantitative and nonradioactive, and has proven to be a reproducible and accurate means of quantifying nucleic acid molecules. The bDNA assay offers several advantages for research and clinical applications; it: provides direct quantification of nucleic acid molecules at physiological levels. Since bDNA assays do not require highly purified nucleic acid preparations, inhibitors of enzyme-dependent amplification techniques are of no concern. allows a wide diversity of specimen types to be used. can be used with reference standards to ensure accuracy for applications requiring absolute quantification, or without standards for applications requiring only assessment of relative changes in nucleic acid concentrations. exhibits a high level of sensitivity (moderately expressed genes can be detected using as few as 50–100 cells). The level of sensitivity can be modulated by altering the number of target-specific extenders to accommodate specific applications. is thoroughly tested for within-lot and between-lot precision to ensure reproducibility (2.2- to 3-fold changes can be discerned as statistically significant).