Directed covalent immobilization of aminated DNA probes on aminated plates.
A new protocol that enables the immobilization of DNA probes on aminated micro-titer plates activated with aldehyde-dextran via an amino group artificially introduced in the 3' end of the oligonucleotide probe is reported in this work. The method is based on the use of hetero-functional-dextran as a long and multifunctional spacer arm covalently attached to an aminated surface capable of immobilizing DNA oligonucleotides. The immobilization occurred only via the amino introduced in the 3' end of the probe, with no implication of the DNA bases in the immobilization, ensuring that the full length of the probe is available for hybridization. These plates having immobilized oligonucleotide probes are able to hybridize complementary DNA target molecules. The tailor-made hetero-functional aldehyde-aspartic-dextran together with the chemical blocking of the remaining primary amino groups on the support using acetic anhydride avoid the nonspecific adsorption of DNA on the surface of the plates. Using these activated plates, (studying the effect of the probe concentration, temperature, and time of the plate activation on the achieved signal), thus, the covalent immobilization of the aminated DNA probe was optimized, and the sensitivity obtained was similar to that achieved using commercial biotin-streptavidin systems. The new DNA plates are stable under very drastic experimental conditions (90% formamide, at 100 degrees C for 30 min or in 100 mM NaOH).
Fuentes
Manuel
M
Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
Mateo
Cesar
C
García
Lucia
L
Tercero
Juan C
JC
Guisán
José M
JM
Fernández-Lafuente
Roberto
R
D000327:Adsorption; D000588:Amines / Q000737:chemistry*; D001483:Base Sequence; D017931:DNA Primers; D015342:DNA Probes
D000588:Amines; D017931:DNA Primers; D015342:DNA Probes