Importance

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Abstract

A method of simultaneous determination of the identity of nucleotide bases at specific positions in nucleic acids of interest, which includes (a) treating a sample containing the nucleic acids of interest to obtain unpaired nucleotide bases spanning the specific positions, if the nucleic acids are not already single stranded; (b) contacting the unpaired nucleotide bases with combinations of various marked oligonucleotide primers each for hybridizing with a stretch of nucleotide bases present in each nucleic acid of interest immediately adjacent the nucleotide base to be identified, so as to form a duplex between the primer and the nucleic acid of interest such that the nucleotide base to be identified is the first unpaired base in the template immediately 5' of the nucleotide base annealed with the 3'-end of the primer in the duplex; (c) contacting the duplex with the reagent which includes an aqueous carrier, and at least one primer extension unit, the primer extension unit including an extension moiety a separation moiety and a detection moiety, with the extension moiety for specifically halting a nucleic acid template dependent, primer extension reaction, in a manner which is strictly dependent on the identity of the unpaired nucleotide base of the template immediately adjacent to, and 3' of, the 3'-end of the primer, with the separation moiety permitting the affinity separation of the primer extension unit from unincorporated, or non-extended, primers, and with the detection moiety enabling the direct or indirect detection of the presence of a primer extension unit the contacting taking place under conditions permitting the base pairing of the complementary extension moiety of the primer extension unit present in the reagent with the nucleotide base to be identified and the occurrence of a template dependent, primer extension reaction to incorporate the extension moiety of the primer extension unit at the 3'-end of the primer, resulting in the extension of the primer by a single unit; (d) removing the non-extended marked primer; (e) determining the presence of a nucleotide alteration; and (f) determining the identity of the extended primers, and therefore the kind of alterations and the complete genotype of the examined nucleic acid, by hybridizing the extended primers to complementary oligonucleotides adhered to a test surface.

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First Claim