Appeal No. 1999-2814
Application No. 08/990,539
27. In a method for determining the nucleotide base sequence of a
nucleic acid molecule, which comprises the steps of:
a) annealing said nucleic acid molecule with a primer molecule
able to hybridize to said nucleic acid molecule;
b) incubating the annealed mixture with four different
deoxynucleoside triphosphates, a polymerase, and a nucleic
acid synthesis terminating agent which terminates synthesis at a
specific nucleotide base; and
c) separating the products of step b);
the improvement comprising detecting the separated products
via a fluorescent metal-ligand complex, wherein when the metal
in each said fluorescent metal-ligand complex is Ru, the
complex is selected from the group consisting of [Ru(2,2’-
bipyridyl)2(1,10-phenanthroline-9-isothiocyanate)]2+, [Ru(4,7-
diphenyl-1,10-phenanthroline)2(4,4’-dicarboxylic acid-2,2’-
bipyridine)]2+, [Ru(4,7-diphenyl-1,10-phenanthroline)2(4-methyl,
4’-carboxylic acid-2,2’-bipyridine)]2+, [Ru (4,7-diphenyl-1,10-
phenanthroline(SO3Na) 2)2(4,4’-dicarboxylic acid-2,2’-
bipyridine)]2+, [Ru(4,7-diphenyl-1,10-phenanthroline(SO3Na) 2)2
(4-methyl,4’-carboxylic acid-2,2’-bipyridine)]2+, and [Ru bis(2,2’-
bipyridyl) (phenanthroline-maleamide)].
The examiner relies on the following references:
Bannwarth, “Bathophenanthroline-Ru(II) complexes as nonradioactive labels for
dideoxy DNA sequencing,” Analytical Biochemistry, Vol. 181, pp. 216-219 (1989)
Zhang et al. (Zhang), “Use of non-cross-linked polyacrylamide for four-color DNA
sequencing by capillary electrophoresis separation of fragments up to 640 bases
in length in two hours,” Analytical Chemistry, Vol. 67, pp. 4589-4593 (1995)
Soper et al. (Soper), “On-Line Fluorescence Lifetime Determina tions in Capillary
Electrophoresis,” Analytical Chemistry, Vol. 67, pp.4358-4365 (1995)
Terpetschnig et al. (Terpetschnig), “Fluorescence polarization immunoassay of a
high-molecular-weight antigen using a long wavelength absorbing and laser
diode-excitable metal-ligand complex,” Analytical Biochemistry, Vol. 240, pp.
54-59 (1996)
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