Importance

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Abstract

A method aimed at the quantification of di- and trinucleotide repeat which includes (a) treating a sample containing the nucleic acids of interest to obtain unpaired nucleotide bases spanning the position of the repeats and flanking regions, if the nucleic acids are not already single stranded; (b) contacting the unpaired nucleotide bases with an oligonucleotide primer capable of hybridizing with a stretch of nucleotide bases present in the nucleic acid of interest preferably 3' of the trinucleotide repeats to be quantified, so as to form a duplex between the primer and the nucleic acid of interest; (c) providing means to ensure that the examined nucleic acid and the oligonucleotide primer are confined to a reaction chamber at all further steps; (d) contacting the duplex with a primer extension unit which is capable of base pairing with the first nucleotide base in the core sequence of the repeats, and a template dependent extension enzyme; (e) eliminating non-incorporated primer extension units; (f) contacting the template primer duplex with a primer extension unit which is capable of base pairing with the second nucleotide base in the core sequence of the repeats, and a template dependent extension enzyme; (g) eliminating non-incorporated primer extension units; (h) contacting the template primer hybrid with a primer extension unit which is capable of base pairing with the third nucleotide base in the core sequence of the repeats; a detection moiety containing, primer extension unit which is capable of base pairing with a nucleotide base 5' of the repeats region, said nucleotide base being the first nucleotide base of a type not included among the nucleotide bases in the core sequence of the trinucleotide repeats; and a template dependent extension enzyme; (i) eliminating non-incorporated primer extension units; (j) detecting for the presence of detection moiety containing primer extension unit; (k) steps (d) to (j) are repeated until detecting said detection moiety; (l) the number of repeats as stated under (k) enables the determination of the number of trinucleotide repeats, therefore enabling determination of the exact repetition number.

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First Claim