Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-19
2002-06-11
Zitomer, Stephanie W. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S018000, C435S287200, C435S288300, C536S023100, C536S024330
Reexamination Certificate
active
06403309
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to compositions and methods for use in screening nucleic acid populations for nucleotide polymorphisms. The methods, referred to generally as ValiGene
SM
Mutation Screening, Peptide-Linked (VGMS-PL) methods, are specifically designed for high-throughput genotype mapping and gene expression analysis of nucleic acids without requiring a PCR amplification step. In particular, the methods of the invention utilize oligonucleotide probes labeled with distinguishable and identifiable labels, (e.g., peptide tags), that are captured on addressable antibody arrays for analysis (e.g., by fluorescence photometry).
2. BACKGROUND OF THE INVENTION
With the advent of genome-wide sequencing efforts, understanding the molecular basis of all genetic diseases may soon be within reach. Single nucleotide polymorphism (SNP) detection analysis is playing an increasingly powerful role in mapping out the underlying genetic basis of many human diseases.
Approximately 1 in every 1000 nucleotides differs between any two copies of the human genome (Cooper, 1996, Hum. Genet. 69:201-205). Some of these genetic variations, or SNPs, lead to differences in the proteins encoded by such genes. Others are “silent”, residing in non-protein coding regions of the genome. Such SNPs are now being used, for example, to diagnose genetic disorders, determine a predisposition to genetic disease, identify or determine the ancestry of a genetic sample, or correlate genetic sequences with phenotypic conditions, such as complex disorders or drug response and toxicity (Risch and Merikangas, 1996, Science 273:1516-1517). This powerful combination of genetic and molecular biological approaches is changing the face of drug development. SNPs have been correlated with Huntington's disease, Alzheimer's disease, and various forms of breast cancer. In the emerging field of pharmacogenomics, specific SNPs are being used to determine and predict a patient's susceptibility to diseases as well as drug toxicity and reponse. Pharmacogenomics can also provide tools to identify new targets for designing drugs and to optimize the use of existing drugs. The hope is that this understanding will ultimately lead to the early diagnosis, prevention, and treatment of genetic diseases.
Single nucleotide polymorphisms can be identified by a number of methods, including DNA sequencing, restriction enzyme analysis, or site-specific hybridization. However, high-throughput genome-wide screening for SNP and mutations requires the ability to simultaneously analyze multiple loci with high accuracy and sensitivity. To increase sensitivity, current high-throughput methods for single nucleotide detection rely on a step that involves amplification of the target nucleic acid sample, usually by the polymerase chain reaction (PCR) (see, e.g., Nikiforov et al., U.S. Pat. No. 5,679,524 issued Oct. 21, 1997; McIntosh et al., PCT publication WO 98/59066 dated Dec. 30, 1998; Goelet et al., PCT publication WO 95/12607 dated May 11, 1995; Wang et al., 1998, Science 280:1077-1082; Tyagi et al., 1998, Nature Biotechnol. 16:49-53; Chen et al., 1998, Genome Res. 8:549-556; Pastinen et al., 1996, Clin. Chem. 42:1391-1397; Chen et al, 1997, Proc. Natl. Acad. Sci. 94:10756-10761; Shuber et al., 1997, Hum. Mol. Gen. 6:337-347; Liu et al., 1997, Genome Res. 7:389-398; Livak et al., Nature Genet. 9:341-342; Day and Humphries, 1994, Annal. Biochem. 222:389-395). However, the fidelity of the PCR technique is limited. Combinations of pairs of PCR primers tend to generate spurious reaction products. Moreover, the number of errors in the final reaction product increases exponentially with the each round of PCR amplification after an error is introduced into a DNA sample. Thus, PCR error can be a substantial drawback when searching for rare variations in nucleic acid populations.
For all of the reasons addressed above, a highly sensitive, highly specific, PCR-free method for high-throughput detection of nucleic acid variations is urgently needed. This invention provides such a method, as described in detail below.
Citation or discussion of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.
3. SUMMARY OF THE INVENTION
The present invention provides methods for detection of single nucleotide polymorphisms (SNPs) and other variations in nucleic acid populations. The methods of the present invention for high-throughput PCR-free screening are used for detection of alterations and polymorphisms as well as for analysis of gene expression, both qualitative and quantitative, directly from cellular total RNA. The methods may be used, for example, to diagnose disorders, determine predisposition to genetic diseases, determine identity or ancestry, or correlate genetic sequences with phenotypic conditions.
In a specific embodiment, the invention relates to methods for efficient, sensitive, high-throughput addressable-array based screens using SNP-specific oligonucleotide probes having distinguishable and identifiable peptide tags to detect polymorphisms in nucleic acid target molecules. First, SNP-specific peptide-linked oligonucleotide probes, comprising distinguishable markers, are hybridized to a target nucleic acid sample. Next, any hybrid molecules formed are captured on high-density addressable antibody arrays and processed by enzymes that recognize and cleave the captured hybrid molecules at mismatched base pairs. Finally, markers present on the cleaved hybrid molecules are then detected and analyzed to identify any polymorphic site(s) within a specific target nucleic acid molecule of interest.
The present methods offer several advantages over the currently available technologies for genotype detection. First, the methods described herein allow detection of genetic variation using minimal amounts of genetic material without requiring a PCR amplification step, avoiding the introduction of new mutations into the sample being tested. In other genotyping methods, a PCR amplification step is typically used to amplify the signal of a given target sequence within a nucleic acid sample to allow detection. In the present invention, it is the signals that are amplified from a number of limited targets. This allows reliable SNP detection using minimal amounts of genetic material from a variety biological sources, such as biopsies of tissue from a patient with a potential genetic disorder. Second, unlike in other methods of genotype mapping, nucleic acid hybridization takes place in solution, eliminating the need to immobilize a hybridization partner. Solution hybridization is more efficient than hybridization with one immobilized partner, resulting in increased efficiency of mismatch detection. Third, the addressable chip array allows a flexible detection system for high-throughput genotype analysis. Multiple SNP sites can be screened simultaneously from a patient or genetic sample, or alternatively, a single SNP site in many different DNA samples, can be tested simultaneously. For example, in one embodiment, detection of multiple polymorphisms in a target nucleic acid sample is possible in a single run. Multiple probes can be individually prepared, each probe having a unique peptide label and a sequence corresponding to a polymorphic site to be detected. Such multiple probes can be hybridized in “batch” with the target nucleic acid sample. In another embodiment, multiple target nucleic acid samples can be screened simultaneously for the presence or absence of a single SNP locus.
Throughout this application reference is made to peptide labels and antibodies for binding said labels. In addition to peptide-antibody combinations, it will be understood by those skilled in the art that any label can be used, in combination with a suitable binding partner. Examples of such labels and binding partners include, but are not limited to, digoxigenin-antidigoxigenin, biotin-streptavidin, ligand (e.g. hormone)-receptor and carbohydrate-lectin combinations.
The term “polymorphism” as
Iris Francois J.-M.
Pourny Jean-Louis
Pennie & Edmonds LLP
ValiGen (US), Inc.
Zitomer Stephanie W.
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