Method of determining the presence and quantifying the number of

Details Method of determining the presence and quantifying the number of Method of determining the presence and quantifying the number of

1994-10-04

1997-07-22

Jones, W. Gary

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 911, 435 912, 435 9152, 536 2433, 935 77, 935 78, C07H 2104, C12P 1934, C12Q 168

Patent

active

056502770

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.

REFERENCES:
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patent: 5378602 (1995-01-01), Polymeropoulos et al.
patent: 5468610 (1995-11-01), Polymeropoulos et al.
Yamamoto et al., Bioch. Biophys. Res. Comm. 182(2):507-513 Jan. 31, 1992.
Jalanko et al., Clin. Chem. 38(1):39-43 1991.
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Kuppuswamy et al., Proc. Natl. Acad. Sci., USA, 88:1143-1147 1991.
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