Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-12-13
2003-02-11
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S024330
Reexamination Certificate
active
06518024
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to detection of mutated nucleic acid and genetic polymorphisms by single base extension analysis. Specifically, the invention relates to single base extension following hybridization of a biological sample comprising a nucleic acid with an oligonucleotide array. In particular, the invention provides apparatus and methods for electronic detection of single base extension of a particular oligonucleotide in an oligonucleotide array after hybridization to a nucleic acid in a biological sample and single base extension thereof.
2. Background of the Invention
The detection of single base mutations and genetic polymorphisms in nucleic acids is an important tool in modern diagnostic medicine and biological research. In addition, nucleic acid-based assays also play an important role in identifying infectious microorganisms such as bacteria and viruses, in assessing levels of both normal and defective gene expression, and in detecting and identifying mutant genes associated with disease such as oncogenes. Improvements in the speed, efficiency, economy and specificity of such assays are thus significant needs in the medical arts.
Ideally, such assays should be sensitive, specific and easily amenable to automation. Efforts to improve sensitivity in nucleic acid assays are known in the prior art. For example, the polymerase chain reaction (Mullis, U.S. Pat. No. 4,683,195, issued Jul. 28, 1987) provides the capacity to produce useful amounts (about 1 &mgr;g) of a specific nucleic acid in a sample in which the original amount of the specific nucleic acid is substantially smaller (about 1 pg). However, the prior art has been much less successful in improving specificity of nucleic acid hybridization assays.
The specificity of nucleic acid assays is determined by the extent of molecular complementary of hybridization between probe and target sequences. Although it is theoretically possible to distinguish complementary targets from one or two mismatched targets under rigorously-defined conditions, the dependence of hybridization on target/probe concentration and hybridization conditions limits the extent to which hybridization mismatch can be used to reliably detect, inter alias, mutations and genetic polymorphisms.
Detection of single base extension has been used for mutation and genetic polymorphism detection in the prior art.
U.S. Pat. No. 5,925,520 disclosed a method for detecting genetic polymorphisms using single base extension and capture groups on oligonucleotide probes using at least two types of di deoxy, chain-terminating nucleotide triphosphate, each labeled with a detectable and distinguishable fluorescent labeling group.
U.S. Pat. No. 5,710,028 disclosed a method of determining the identity of nucleotide bases at specific positions in nucleic acids of interest, using detectably-labeled chain-terminating nucleotides, each detectably and distinguishably labeled with a fluorescent labeling group.
U.S. Pat. No. 5,547,839 disclosed a method for determining the identity of nucleotide bases at specific positions in a nucleic acid of interest, using chain-terminating nucleotides comprising a photo removable protecting group.
U.S. Pat. No. 5,534,424 disclosed a method for determining the identity of nucleotide bases at specific positions in a nucleic acid of interest, using each of four aliquots of a target nucleic acid annealed to an extension primer and extended with one of four chain-terminating species, and then further extended with all four chain-extending nucleotides, whereby the identity of the nucleotide at the position of interest is identified by failure of the primer to be extended more that a single base.
U.S. Pat. No. 4,988,617 disclosed a method for determining the identity of nucleotide bases at specific positions in a nucleic acid of interest, by annealing two adjacent nucleotide primers to a target nucleic acid and providing a linking agent such as a ligase that covalently links the two oligonucleotides to produce a third, combined oligonucleotide only under circumstances wherein the two oligonucleotides are perfectly matched to the target nucleic acid at the 3′ extent of the first oligonucleotide and at the 5′ extent of the second oligonucleotide.
U.S. Pat. No. 4,656,127 disclosed a method for determining the identity of nucleotide bases at specific positions in a nucleic acid of interest, using primer extension with a chain-terminating or other nucleotide comprising an exonuclease-resistant linkage, followed by exonuclease treatment of the plurality of extension products to detect the resistant species therein. One common feature in this prior art is that single base extension has been detected by incorporation of fluorescent labels into the extended nucleic acid species.
A significant drawback of single base extension methods based on fluorescent label detection is the need for expensive and technically-complex optical components for detecting the fluorescent label. Although fluorescent probes used in such methods impart an adequate level of discrimination between extended and unextended positions in an oligonucleotide array, these methods typically require detection of up to four different fluorescent labels, each having a unique excitation and fluorescence emission frequency. As a consequence of these properties, such assay systems must be capable of producing and distinguishing light at all of these different excitation and emission frequencies, significantly increasing the cost and complexity of producing and operating apparatus used in the practice thereof.
An alternative method for detecting a target nucleic acid molecule is to use an electrochemical tag (or label) such as a redox moiety in combination with an electrochemical detection means such as cyclic voltammetry.
U.S. Pat. No. 5,591,578 provides for the selective covalent modification of nucleic acids with redox-active moieties such as transition metal complexes of specifically-claimed transition metals, wherein the complexes are covalently linked to a ribose sugar comprising the ribose-phosphate backbone. The resulting complexes are capable of transferring electrons over very large distances at extremely fast rates.
U.S. Pat. No. 5,705,348, related to U.S. Pat. No. 5,591,578, encompasses generally selective covalent modification of nucleic acids with redox-active moieties such as transition metal complexes, wherein the transition metals are generically-claimed.
U.S. Pat. No. 5,770,369, related to U.S. Pat. No. 5,591,578 discloses electron donor and acceptor moieties that are not redox proteins.
U.S. Pat. No. 5,780,234 369, related to U.S. Pat. No. 5,591,578, discloses methods wherein two single stranded nucleic acid are used to hybridize to two different domains of the target sequence.
In addition, disclosure of similar methods for detecting biological molecules such as DNA and proteins can be found in Ihara et al., 1996
, Nucleic Acids Res
. 24: 4273-4280; Livache et al., 1995
, Synthetic Metals
71: 2143-2146; Hashimoto, 1993
, Supramolecular Chem
. 2: 265-270; Millan et al., 1993, Anal. Chem. 65: 2317-2323.
However, most of the electrochemical tag-dependent methods known in the prior art require hybridization of the probe/target in the presence of a redox intercalator. Electrochemical detection based on redox intercalators are generally not as reproducible as redox tags that are covalently bound to an incorporated moiety. Redox intercalator methods are exceedingly dependent on washing conditions to remove excess label while not reducing the actual signal. As a consequence, false positives are often obtained using these methods. The specificity of redox intercalator methods is often much worse than can be achieved with covalently-bound redox tags.
There remains a need in this art for simple, economical, and efficient ways to detect single base extension products of nucleic acid assays for detecting mutation and genetic polymorphisms in biological samples containing a nucleic acid of interest.
SUMMARY OF THE INVENTIO
Choong Vi-En
Gallagher Sean
Maracas George
Shi Song
Brusca John S.
Dorsey & Whitney LLP
Kim Young
Motorola Inc.
Silva Robin M.
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