METHOD OF DETERMINING THE PRESENCE AND QUANTIFYING THE NUMBER OF DI- AND TRINUCLEOTIDE REPEATS AND AN INSTRUMENT AND KITS THEREOF

摘要

A method aimed at the quantification of di- and trinucleotide repeatwhich includes (a) treating a sample containing the nucleic acids of interest toobtain unpaired nucleotide bases spanning the position of the repeats andflanking regions, if the nucleic acids are not already single stranded; (b)contacting the unpaired nucleotide bases with an oligonucleotide primer capableof hybridizing with a stretch of nucleotide bases present in the nucleic acid ofinterest preferably 3' of the trinucleotide repeats to be quantified, so as to form aduplex between the primer and the nucleic acid of interest; (c) providing meansto ensure that the examined nucleic acid and the oligonucleotide primer areconfined to a reaction chamber at all further steps; (d) contacting the duplexwith a primer extension unit which is capable of base pairing with the firstnucleotide base in the core sequence of the repeats, and a template dependentextension enzyme; (e) eliminating non-incorporated primer extension units; (f)contacting the template primer duplex with a primer extension unit which iscapable of base pairing with the second nucleotide base in the core sequence ofthe repeats, and a template dependent extension enzyme; (g) eliminatingnon-incorporated primer extension units; (h) contacting the template primer hybridwith a primer extension unit which is capable of base pairing with the thirdnucleotide base in the core sequence of the repeats; a detection moietycontaining, primer extension unit which is capable of base pairing with anucleotide base 5' of the repeats region, said nucleotide base being the firstnucleotide base of a type not included among the nucleotide bases in the coresequence of the trinucleotide repeats; and a template dependent extensionenzyme; (i) eliminating non-incorporated primer extension units; (j) detectingfor the presence of detection moiety containing primer extension unit; (k) steps(d) to (j) are repeated until detecting said detection moiety; (l) the number ofrepeats as stated under (k) enables the determination of the number oftrinucleotide repeats, therefore enabling determination of the exact repetitionnumber.