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Branched DNA assay

In biology, a branched DNA assay is a test for specific nucleic acid chains, and is typically used to detect retroviruses such as HIV.[1] However, the assay can be used to detect and quantitate other types of RNA or DNA target. In the assay, branched DNA is mixed with a sample to be tested. The detection is done using a non-radioactive method and does not require preamplification of the nucleic acid to be detected. The assay entirely relies on hybridization. Enzymes are used to indicate the extent of hybridization but are not used to manipulate the nucleic acids. Thus, small amounts of a nucleic acid can be detected and quantified without a reverse transcription step (in the case of RNA) and/or PCR. The assay can be run as a "high throughput assay", unlike quantitative Northern-blotting or the RNAse-protection assay, which are labor-intensive and thus difficult to perform on a large number of samples. The other major high throughput technique employed in the quantitation of specific RNA molecules is quantitative PCR, after reverse transcription of the RNA to cDNA.

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.[2] Despite the fact that the starting material is not preamplified, the assays are extraordinarily sensitive. Newer bDNA assays can detect less than 100 copies of HIV-RNA per mL of blood.

See also

* Dendrimer


1. ^ Murphy, D. G.; Gonin, P.; Fauvel, M. (1999). "Reproducibility and Performance of the Second-Generation Branched-DNA Assay in Routine Quantification of Human Immunodeficiency Virus Type 1 RNA in Plasma". Journal of Clinical Microbiology (American Society for Microbiology) 37 (3): 812–814. doi:0095-1137/99/$04.00+0. PMID 9986862. PMC PMC84566. http://jcm.asm.org/cgi/content/abstract/37/3/812.
2. ^ Collins, M. L.; Irvine, B.; Tyner, D.; Fine, E.; Zayati, C.; Chang, C.; Horn, T.; Ahle, D.; Detmer, J.; Shen, L. P.; Kolberg, J.; Bushnell, S.; Urdea, M. S.; Ho, D. D. (1997). "A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml". Nucleic Acids Research 25 (15): 2979–2984. doi:10.1093/nar/25.15.2979. PMID 9224596. PMC PMC146852. http://nar.oxfordjournals.org/cgi/content/full/25/15/2979.

External links

* MeSH Branched+DNA+Assay


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