Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-26
2002-03-26
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C600S345000
Reexamination Certificate
active
06361951
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to nucleic acid hybridization and sequencing, and particularly to methods of qualitatively and quantitatively detecting nucleic acid hybridization and to methods of nucleic acid sequencing.
BACKGROUND OF THE INVENTION
The detection of individual DNA sequences in heterogenous samples of DNA provides a basis for identifying genes, DNA profiling, and novel approaches to DNA sequencing. One approach to DNA hybridization detection involves the use of surface bound DNA sequences which can be assayed using an analytical response that indicates hybridization of the surface-bound oligomer to a sequence in the heterogeneous sample. These analytical methods generally involve laser-induced fluorescence arising from a covalently attached label on the target DNA strand, which is not sensitive to single-base mismatches in the surface-bound duplex. For example, U.S. Pat. Nos. 5,143,854 and 5,405,783 to Pirrung et al.; Fodor, et al.,
Nature
364:555 (1993); Bains,
Angew. Chem.
107:356 (1995); and Noble,
Analytical Chemistry
67(5):201A (1995) propose surfaces or “chips” for this application. In an alternate method, proposed by Hall, et al.,
Biochem. and Molec. Bio. Inter.
32(1):21 (1994), DNA hybridization is detected by an electrochemical method including observing the redox behavior of a single stranded DNA as compared to a double stranded DNA. This technique is also not sensitive to single-base mismatches in the DNA sample. Techniques for detecting single-base mismatches include enzymatic or chemical cleavage studies, such as those proposed in U.S. Pat. No. 5,194,372 to Nagai et al. However, these techniques are disadvantageous inasmuch as they require more time and separation technology.
U.S. Pat. No. 5,312,527 to Mikkelson et al. describes a voltammetric sequence-selective sensor for detecting target nucleic acid in which a double-stranded nucleic acid is contacted to a redox-active complex. The complex binds non-specifically to the double-stranded DNA. Because the complex itself is the redox-active compound that provides a voltammetric signal, the complex does not function in a catalytic manner without the addition of an enzyme.
U.S. Pat. No. 4,840,893 to Hill et al. describes an electrochemical assay for nucleic acids in which a competitive binding event between a ligand and an antiligand is in turn detected electrochemically.
Accordingly, there remains a need in the art for a method of detecting DNA hybridization, including a method of detecting single-base pair mismatches, which is both rapid and sensitive, and which can be rapidly applied on-line.
SUMMARY OF THE INVENTION
In general, the present invention provides a method of detecting a nucleic acid that contains at least one preselected base (e.g., adenine, guanine, 6-mercaptoguanine, 8-oxo-guanine, and 8-oxo-adenine). The method comprises (a) reacting the nucleic acid with a transition metal complex capable of oxidizing the preselected base in an oxidation-reduction reaction; (b) detecting the oxidation-reduction reaction; and (c) determining the presence or absence of the nucleic acid from the detected oxidation-reduction reaction at the preselected base. Depending on the particular embodiment of the method and the particular object desired, the method may optionally include the step of contacting the nucleic acid with a complementary nucleic acid to form a hybridized nucleic acid.
As a first aspect, the present invention provides a method of detecting DNA hybridization. The method includes (a) contacting a DNA sample with an oligonucleotide probe to form a hybridized DNA, (b) reacting the hybridized DNA with a transition metal complex capable of oxidizing a preselected base in the oligonucleotide probe in an oxidation-reduction reaction where the oligonucleotide probe has at least one of the preselected bases, (c) detecting the oxidation-reduction reaction, (d) determining the presence or absence of hybridized DNA from the detected oxidation-reduction reaction at the preselected base. As discussed in detail below, the step of detecting the oxidiation-reduction reaction may, in general, be carried out by measuring electron flow from the preselected base.
As a second aspect, the present invention provides another method of detecting DNA hybridization. The method includes (a) contacting a DNA sample with an oligonucleotide probe to form a hybridized DNA, (b) reacting the hybridized DNA with a transition metal complex capable of oxidizing a preselected base in the oligonucleotide probe in an oxidation-reduction reaction, where the oligonucleotide probe has at least one of the preselected bases, (c) detecting the oxidation-reduction reaction, (d) measuring the reaction rate of the detected oxidation-reduction reaction, (e) comparing the measured reaction rate to the oxidation-reduction reaction rate of the transition metal complex with a single-stranded DNA, and then (f) determining whether the measured reaction rate is essentially the same as the oxidation-reduction reaction rate of the transition metal complex with single-stranded DNA.
As a third aspect, the present invention provides an apparatus for detecting DNA hybridization. The apparatus includes (a) a plurality of DNA sample containers, (b) sample handling means for carrying the plurality of DNA sample containers, (c) an oligonucleotide probe delivery means for delivering the oligonucleotide probe to each of the DNA sample containers, (d) a transition metal complex delivery means for delivering the transition metal complex to each of the plurality of DNA sample containers, and (e) an oxidation-reduction reaction detector for detecting an oxidation-reduction reaction.
As a fourth aspect, the present invention provides a second apparatus for detecting DNA hybridization. The apparatus includes (a) a DNA sample container, (b) an oligonucleotide probe delivery means for delivering a plurality of oligonucleotide probes to the DNA sample container, (c) a transition metal complex delivery means for delivering the transition metal complex to the DNA sample container, and (d) an oxidation-reduction reaction detector for detecting an oxidation-reduction reaction.
As a fifth aspect, the present invention provides a method of sequencing DNA. The method includes (a) contacting a DNA sample with an oligonucleotide probe to form a hybridized DNA, where the oligonucleotide probe includes a preselected synthetic base having a unique oxidation potential, (b) reacting the hybridized DNA with a transition metal complex capable of oxidizing the preselected synthetic base in the oligonucleotide probe in an oxidation-reduction reaction, where the oligonucleotide probe has a predetermined number of the preselected synthetic bases, (c) detecting the oxidation-reduction reaction, (d) measuring the reaction rate of the detected oxidation-reduction reaction, and (e) identifying the base paired with the preselected synthetic base.
The foregoing and other aspects of the present invention are explained in detail in the detailed description set forth below.
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Johnston Dean H.
Kim Jin-heung
Loomis Carson R.
Napier Mary E.
Sistare Mark F.
Chakrabarti Arun
Myers Bigel & Sibley & Sajovec
The University of North Carolina at Chapel Hill
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