Branched DNA assay

Method

A branched DNA assay begins with a dish or some other solid support (e.g., a plastic dipstick). The dish is peppered with small, single stranded DNA molecules (or chains) that stick out into the solution. These are known as capture probe DNA molecules. Next, an extender DNA molecule is added. Each extender has two domains; one that hybridizes to the capture DNA molecule and one that sticks out above the surface. The purpose of the extender is two-fold. First, it creates more available surface area for target DNA molecules to bind, and second, it allows the assay to be easily adapted to detect a variety of target DNA molecules.

Once the capture and extender molecules are in place and they have hybridized, the sample can be added. Target molecules in the sample will bind to the extender molecule. This results in a base peppered with capture probes, which are hybridized to extender probes, which in turn are hybridized to target molecules.

At this point, signal amplification takes place. A label extender DNA molecule is added that has two domains (similar to the first extender). The label extender hybridizes to the target and to a preamplifier molecule. The preamplifier molecule has two domains. First, it binds to the label extender and second, it binds to the amplifier molecule. An example amplifier molecule is an oligonucleotide chain bound to the enzyme alkaline phosphatase.

Diagrammatically, the process can be resembled as

Base → Capture Probe → Extender → Target → label extender → pre-amplifier → amplifier

Process

Several different short single-stranded DNA molecules (oligonucleotides) are used in a branched DNA-assay. The capture and capture-extender oligonucleotide bind to the target nucleic acid and immobilize it on a solid support. The label oligonucleotide and the branched DNA then detects the immobilized target nucleic acid. The immobilization of the target on a solid support makes extensive washing easier, which reduces false positive results. After binding of the target to the solid support it can be detected by branched DNA which is coupled to an enzyme (e.g. alkaline phosphatase). The branched DNA binds to the sample nucleic acid by specific hybridization in areas which are not occupied by capture hybrids. The branching of the DNA allows for very dense decorating of the DNA with the enzyme, which is important for the high sensitivity of the assay. The enzyme catalyzes a reaction of a substrate which generates light (detectable in a luminometer). The amount of light emitted increases with the amount of the specific nucleic acid present in the sample. The design of the branched DNA and the way it is hybridized to the nucleic acid to be investigated differs between different generations of the bDNA assay.

Generations

The first-generation bDNA assay directly uses a branched DNA molecule attached to many "alkaline phosphatase probe" molecules (amplifier), which binds to the label extender and then to the target molecule. It could accurately quantify between ~10 000 and 10 000 000 molecules.

The second-generation assay adds a "preamplifier" molecule to further enhance the sensitivity. The preamplifier binds the label extender and several copies of the amplifier, making the signal much stronger. It could detect down to 500 molecules.

The third-generation or "system 8" assay uses isoguanine and isocytosine residues to prevent non-specific hybridization between the amplifier and preamplifier and the target: these parts are supposed to only bind the label extender, but when they are made too long they also bind the target causing false positives. By preventing non-specific hybridization, longer versions of these two molecules can be used, allowing for more amplification of the signal and a detection limit of 100 copies of HIV-RNA per mL of blood. It was developed under Chiron Diagnostics in 1997 and was owned by Bayer Diagnostics as of 2005–2006 as the "Quantiplex" or "VERSANT" technology. As of 2025 the "VERSANT" brand appears to be owned by Siemens.

There is not a universally agreed-upon definition of a fourth-generation assay, but this does not mean improvements have ceased to occur. For example, a 2005 patent increases the specificity enough for use in single nucleotide polymorphism detection.