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Conjugation & Surface Attachment Applications

Introduction to Conjugation & Surface Attachment Conjugation & Surface Attachment Applications Conjugation & Surface Attachment Design/Protocol Conjugation & Surface Attachment Literature Order Online

Conjugation & Surface Attachment Applications

One of the most common applications for amino-modified oligos is for conjugation to fluorescent dyes through an NHS ester. Many dyes with desirable properties (for example, absorption/emission wavelengths or high fluorescence intensity) are not available as phosphoramidites, but only as NHS esters. Rhodamine-based dyes and Alexa dyes are two common examples. Certain haptens such as digoxigenin also require conjugation to amino-labeled oligos via an NHS ester, due to lack of a phosphoramidite for it. Amino-labeled oligos also widely used in the manufacture of DNA microarrays used in gene expression studies, with the oligos covalently immobilized to a glass or other silicon-based flat surface through the amine (1). Thiol-modified oligos can be conjugated to a variety of fluorescent or non-fluorescent molecules; the conjugation chemistry here is typically either maleimide or iodoacetamide-based (2). The orthogonality of the corresponding conjugation chemistries for amine and thiol groups allows for the synthesis of oligos with novel combinations of modifications. Thiol-modified oligos can also be immobilized to glass slides, gold flat surfaces or microspheres for use in DNA microarray, nanoelectronic and DNA sensor-based applications (3, 4). However, because the chemical linkage between a single thiol group and gold is somewhat labile, the DTPA (dithiolphosphoramidite) modification permits multiple tethering of an oligo to a gold surface. Incorporation of three units of DTPA has been shown to provide maximum stability (5).

References

(1) Immobilization Chemistry. in Immobilization of DNA on Chips II, C. Whittman (Vol. Ed.). Springer-Verlag, Berlin, Heidelberg (2005): 51-53.
(2) Connolly, B.A., Rider, P. Chemical synthesis of oligonucleotides containing a free sulphydryl group and subsequent attachment of thiol specfic probes. Nucleic Acids Res. (1985), 13: 4485-4502.
(3) Roger, Y-H., Jiang-Baucom, P., Huang, Z-J., Bogdanov, V., Anderson, S., Boyce-Jacino, M.T. Immobilization of oligonucleotides onto a glass support via disulfide bonds: A method for preparation of DNA microarrays. Anal. Biohem. (1999), 266: 23-30.
(4) Ackerson, C.J., Sykes, M.T., Kornberg, R.D. Defined DNA/nanoparticle conjugates. Proc. Natl. Acad. Sci. USA (2005), 102: 13383-13385.
(5) Li, Z., Jin, R., Mirkin, C.A., Letsinger, R.L. Multiple thiol-anchor capped DNA-gold nanoparticle conjugates Nucleic Acids Res. (2002), 30: 1558-1562.
(6) Guo, Z., Guilfoyle, R.A., Thiel, A.J., Wang, R., Smith, L.M. Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports. Nucleic Acids Res. (1994), 22: 5456-5465.
(7) Yang, L., Tran, D.K., Wang, X. BADGE, Beads Array for the Detection of Gene Expression, a high-throughput diagnostic bioassay. Genome Res. (2001), 11: 1888-1898.
(8) Defoort, J.P. Simulataneous detection of multiplex-amplified human immunodeficiency virus type 1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA using a flow cytometer microsphere-based hybridization assay. J. Clin. Microbiol. (2000), 39: 131-140.

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