Construction of oligonucleotide arrays on a glass surface using a heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA).
A rapid method for construction of oligonucleotide arrays on a glass surface, using a novel heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA), has been described. The heterobifunctional reagent, NTMTA, carries two different thermoreactive groups. The triethoxysilyl group on one end is specific towards silanol functions on the virgin glass surface, while the trifluoroethanesulfonyl (tresyl) group on the other end of the reagent reacts specifically with aminoalkyl- or mercaptoalkyl- functionalized oligonucleotides. Immobilization of oligonucleotides on a glass surface has been realized via two routes. In the first one (A), 5'- aminoalkyl- or mercaptoalkyl-functionalized oligonucleotides were allowed to react with NTMTA to form a oligonucleotide-triethoxysilyl conjugate which, in a subsequent reaction with unmodified (virgin) glass microslide, results in surface-bound oligonucleotides. In the second route (B), the NTMTA reagent reacts first with a glass microslide whereby it generates trifluoroethanesulfonate ester functions on it, which in a subsequent step react with 5'-aminoalkyl or mercaptoalkyl oligonucleotides to generate support-bound oligonucleotides. Subsequently, the oligonucleotide arrays prepared by both routes were analyzed by hybridization experiments with complementary oligonucleotides. The constructed microarrays were successfully used in single and multiple nucleotide mismatch detection by hybridizing these with fluorescein-labeled complementary oligonucleotides. Further more, the proposed method was compared with the existing methods with respect to immobilization efficiency of oligonucleotides.
Kumar
P
P
Nucleic Acids Research Laboratory, Institute for Genomics and Integrative Biology, Mall Road, Delhi University Campus, Delhi 110 007, India.
Choithani
Jyoti
J
Gupta
K C
KC
D020137:Base Pair Mismatch; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D005453:Fluorescence; D005898:Glass; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011437:Propylamines / Q000737:chemistry*; D012680:Sensitivity and Specificity; D012821:Silanes / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors
D003432:Cross-Linking Reagents; C487481:N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine; D009841:Oligonucleotides; D011437:Propylamines; D012821:Silanes