Colourimetric detection is one of the oldest diagnostic techniques. This is based on the interaction of an enzyme e.g. HRP interacting with a substrate. This also requires some form of capture of the target with a hapten labelled probe and the duplex captured using an affinity column or matrix loaded with a suitable protein or antibody. Examples of haptens are biotin, DNP and DIG, the most commonly used being biotin in conjunction with streptavidin or avidin. The enzyme labelled oligo then hybridises to another part of the immobilised target and treatment with the substrate produces a distinctive colour.
The uses of avidin-biotin technology are diverse.1 Applications include the detection of proteins by non-radioactive immunoassays, cytochemical staining, cell separation, isolation of nucleic acids, detection of specific DNA/RNA sequences by hybridisation, and probing of conformational changes in ion channels.
Many of these applications require the use of oligos containing biotin at one or more positions. The availability of functional biotin, in turn, provides the opportunity for immobilisation on pre-coated solid surfaces.2 An extension of this technology using the photocleavable biotin product (2122) is described in Photo-Cleavable Modifiers and Their use with Oligonucleotides.
Several different reagents are available for labelling nucleic acids with biotin. Choosing the right one will depend largely on the position within the oligonucleotide requiring to be labelled. Biotin-CE Phosphoramidite (2140) is based on a 1,3-diol structure where one hydroxyl is protected with DMTr and the other is the phosphoramidite, hence it can be used for adding multiple biotins to either the 3’, or 5’ end of an oligonucleotide. It has been suggested that this property could be exploited in the development of diagnostic probes, in applications such as ELISA, in which signal amplification is often beneficial. This has been shown using in situ hybridisation studies where three biotins at either end of the oligo gives the optimal signal.3
Biotin-TEG-CE Phosphoramidite (2132) can be used in a similar way to 2140 for adding biotin to the 3’- and 5’- ends of an oligo. This phosphoramidite also has an extended 15 atom mixed polarity spacer arm based on a triethylene glycol linker. The benefits of an extended spacer arm separating the biotin function from the rest of the oligo may be seen in applications where possible steric hindrance effects could be reduced as a result, e.g. when dual-labelling with bulky reporter molecules, such as haptens, dyes, or enzymes. Note the 1,2-diol arrangement makes cleavage during deprotection possible therefore it is advisable to keep the 5’-DMTr group on until after deprotection.
5’-Biotin-CE Phosphoramidite (2109) can also be used for adding biotin to an oligo, but only to the 5’-end.4 The DMTr protection on the N1 of biotin prevents branching during coupling. The DMTr group can, however, be used to assist in reverse-phase cartridge and HPLC purification although biotin is hydrophobic enough to obtain good separation of biotin labelled oligos (DMT OFF) and unlabelled oligos.
The addition of biotin internally within an oligonucleotide sequence is achieved using Biotin-dT-CE Phosphoramidite (2067), where any suitable dT position within the sequence can be replaced with biotin-dT. The tert-butylbenzoyl group, used to increase solubility and to protect the biotin, is removed in the ammonium hydroxide deprotection step.
Finally, the direct labelling of the 3’-end of an oligonucleotide sequence with biotin is also possible and is routinely achieved using 3’-Biotin-TEG CPG (2353), which incorporates biotin at the first step in the synthesis process.
- See for example: (a) Avidin-Biotin Technology, M. Wilchek and E.A. Bayer (Eds.), in Methods in Enzymology, J.N. Abelson and M.I. Simon (Series Eds.), Volume 184, 671pp, Academic Press, 1990; [Summary] (b) The biotin-(strept)avidin system: Principles and applications in biotechnology, E.P. Diamandis and T.K. Christopoulos, Clinical Chem., 37, 625-636, 1991. [Full text PDF]
- See for example: Electrochemical detection of non-labelled oligonucleotide DNA using biotin-modified DNA(ss) on a streptavidin-modified gold electrode, J.W. Park, H.-Y. Lee, J.M. Kim, R. Yamasaki, T. Kanno, H. Tanaka, H. Tanaka and T. Kawai, J. Bioscience and Bioengineering, 97, 29-32, 2004. [Abstract]
- A comparative study of digoxigenin, 2,4-dinitrophenyl, and alkaline phosphatase as deoxyoligonucleotide labels in non-radioisotopic in situ hybridisation, S.J. Harper, E. Bailey, C.M. McKeen, A.S. Stewart, J.H. Pringle, J. Feeholly and T. Brown, J. Clinical Pathology, 50, 686-690, 1997. [Full text PDF]
- For a recent diagnostic application see: Detection and differentiation of Plasmodium species by polymerase chain reaction and colorimetric detection in blood sample of patients with suspected malaria, D.M. Whiley, G.M. LeCornec, A. Baddeley, J. Savill, M.W. Syrmis, I.M. Mackay, D.J. Siebert, D. Burns, M. Nissen and T.P. Sloots, Diagnostic Microbiology and Infectious Disease, 49, 25-29, 2004. [Abstract]