Branched DNA assay

In biology, a branched DNA assay is a test for specific nucleic acid chains typically used to detect Retroviruses such as HIVcite journal
author = Murphy, D. G.; Gonin, P.; Fauvel, M.
year = 1999
title = Reproducibility and Performance of the Second-Generation Branched-DNA Assay in Routine Quantification of Human Immunodeficiency Virus Type 1 RNA in Plasma
journal = Journal of Clinical Microbiology
volume = 37
issue = 3
pages = 812–814
publisher = American Society for Microbiology
doi = 0095-1137/99/$04.00 0
url = http://jcm.asm.org/cgi/content/abstract/37/3/812
pmid = 9986862
pmc = PMC84566] . However, the assay can be used to detect and quantitate other RNA or DNA, too. In the assay, Branched DNA is mixed with a sample to be tested. The detection is encompassed by a non-radioactive method and does not require preamplification of the nucleic acid to be detected. The assay entirely relies on hybridisation as principle. Enzymes are used to indicate the extent of hybrdisation 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 is capable to be run as a "high througput assay" which differs from quantitative Northern-blotting or the RNAse-protection assay, which are labor-intense and thus difficult to perform on a large number of samples. The main other high throughput technique employed in the quantitation of specific RNA-molecules is quantitative PCR after reverse transcription of the RNA to cDNA.

Several different single-stranded short DNA-molecules (oligonucleotides) are used in a branched DNA-assay. These function to capture the nucleic acid to be quantified and immobilize it on a solid support (capture and capture-extender) and to detect the nucleic acid (label oligonucleotide and branched DNA). The immobilization of the nucleic acid to be detected on a solid support by specific hybridisation allows for washing and is part of the assay strategy to minimize false positive results. After binding of the nucleic acid to be investigated 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 hybridisation 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.cite journal
author = 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.
year = 1997
title = A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml
journal = Nucleic Acids Research
volume = 25
issue = 15
pages = 2979–2984
doi = 10.1093/nar/25.15.2979
url = http://nar.oxfordjournals.org/cgi/content/full/25/15/2979
pmid = 9224596
pmc = PMC146852] Despite of 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.

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