Branched DNA Method

The branched DNA method (bDNA)63,64 is carried out in microwells and begins with the addition of a lysis buffer to a small volume of serum, plasma, or culture supernatant containing cells or virus. The lysis reagent contains detergent to release target nucleic acid, inhibitors to prevent target degradation, and multiple capture extenders (oligonucleotides) that hybridize to specific areas of the target RNA or DNA. In the case of the HIV bDNA assay, the capture extenders hybridize to multiple sequences in the pol gene. A common sequence on the capture extenders interacts with capture probes immobilized on the surface of 96-microwell plates, thereby anchoring the target nucleic acid to the plate.

Multiple target probes are added that hybridize to different, conserved sequences on the target RNA or DNA. In the HIV bDNA assay, more than 80 target probes covering a large portion of the 3000 bp of the pol gene are used. The target probes contain key sequences that form the foundation for signal amplification, accomplished via the sequential addition of preamplifier (complementary to a region of the target probes), amplifier (complementary to a region of the preamplifier molecule), and alkaline-phosphatase-modified label probes (complementary to portions of the amplifier molecule).

Preamplifier, amplifier, and label probes, as well as the preamplifier region of the binding probes, contain the non-natural nucleotides 5-methyl-2'-deoxyisocytidine (isoMeC) and 2'-deoxyisoguanosine (isoG). These isomers of natural bases can participate in Watson-Crick base pairing with each other but not with cytosine or guanine residues in probes or in DNA or RNA sequences. Incorporation of non-natural bases into the synthetic probe molecules increases the specificity of hybridization by decreasing nonspecific probe interactions, and increases the sensitivity of the assay since higher concentrations of probes can be used.

The series of probes results in formation of large hybridization complexes on the target RNA or DNA. For example, if each hybridization step was 100% efficient in the HIV bDNA assay, each target molecule would be labeled with more than 10,000 alkaline phosphatase molecules. Addition of dioxetane substrate for the alkaline phos-phatase results in steady-state chemiluminescence. The luminescent signal is proportional to the amount of target RNA or DNA present in the sample. The amount of target RNA or DNA in a specimen may be calculated by interpolation from a standard curve generated by signals produced from calibrators that contain known concentrations of the specific viral, bacterial, or cellular RNA or DNA. A schematic of this technology is shown in Figure 2-7.

Examples of Applications of bDNA method

1. HIV quantitation65,66

2. Hepatitis B virus (HBV) quantitation67-70

3. HCV quantitation71,72

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