Appeal No. 2001-1880
Application No. 09/273,835
directed to a method for determining the nucleotide composition of an oligonucleotide
which comprises the steps of:
(a) incorporating a stable, isotope labeled form of one of the four nucleotide units
of an oligonucleotide into the oligonucleotide under investigation in place of the ordinary
nucleotide therein, the other three types of nucleotides in the oligonucleotide being
unlabeled;
(b) measuring the mass peak of the unlabeled oligonucleotide using mass
spectrometry;
(c) measuring the mass peak of the labeled oligonucleotide using mass
spectrometry;
(d) obtaining the magnitude of the mass shift between the labeled
oligonucleotide and the unlabeled oligonucleotide, whereby the number of isotope-
labeled nucleotides in the oligonucleotide under investigation is determined; and
(e) comparing the number of isotope labeled nucleotides with the number of that
type of nucleotide in a reference oligonucleotide.
“By incorporating stable, isotope-labeled nucleotides into oligonucleotides, 'mass
tags' are introduced into PCR products, that is, the substitution of any or all of 13C for
12C, 15N for 14N, and 2H for 1H leads to a mass change of the oligomer.” Specification,
page 6. With the presence of only one type (A,T, C or G) of labeled nucleotide in an
oligomer, the overall mass change of the oligomer corresponds to the number of the
labeled nucleotides in the oligomer, the other three types of nucleotides remaining
unlabeled with their masses unchanged. Specification, pages 6-7. The specification
indicates that “stable isotope labeling requires cells to be grown on a minimal medium
containing 99% (15NH4)2SO4 or 15NH4Cl as the sole source of nitrogen for 15N labeling,
and/or 99% 13CH3OH or sodium [1,2-13C2 99%] acetate as the sole carbon source for
13C labeling, and/or 99% 2H2O as the sole source of deuterium for 2H labeling of DNA.”
Specification, page 8.
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