Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Biological or biochemical
Reexamination Certificate
2000-02-10
2002-09-17
Brusca, John S. (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Biological or biochemical
C435S006120, C536S024300, C536S025400, C536S025500
Reexamination Certificate
active
06453244
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a chromatographic method for the detection or analysis of polymorphisms in nucleic acids, and particularly to denaturing high performance liquid chromatography for such uses.
BACKGROUND OF THE INVENTION
Approximately 4,000 human disorders are attributed to heritable genetic causes. Hundreds of genes responsible for various disorders have been mapped, and sequence information is being accumulated rapidly. A principal goal of the Human Genome Project is to find all genes associated with each disorder.
The most reliable diagnostic test for any specific genetic disease (or predisposition to a particular disease) is the identification of polymorphic variations in DNA sequence in affected cells that result in altered gene function and/or expression levels. In addition, certain polymorphic variations that are associated with predispositions for disorders, e.g., alleles that are associated with disease such as certain forms of cancer or Alzheimer's disease, may allow prophylactic measure to be taken to help reduce or reverse the risk imposed by the polymorphic allele. Furthermore, responses to specific medications may depend on the presence of polymorphisms, making people with a particular polymorphism a better candidate for a medication than those not possessing the polymorphism. These and other reasons provide a great impetus for developing DNA or RNA screening as a practical tool for medical diagnostics.
Genetic polymorphisms and mutations can manifest themselves in several forms, such as point polymorphisms or point mutations where a single base is changed to one of the three other bases, deletions where one or more bases are removed from a nucleic acid sequence and the bases flanking the deleted sequence are directly linked to each other, insertions where new bases are inserted at a particular point in a nucleic acid sequence adding additional length to the overall sequence, and expansions and reductions of repeating sequence motifs. Large insertions and deletions, often the result of chromosomal recombination and rearrangement events, can lead to partial or complete loss of a gene. Of these forms of polymorphism, in general point polymorphisms are the most difficult to detect because they represent the smallest degree of molecular change.
The most definitive screening method to identify and determine polymorphisms such as SNPs in a nucleic acid requires determining the actual base sequence (Maxam and Gilbert, 1977; Sanger et al., 1977). Although such a method is the most accurate, it is also the most expensive and time consuming method. Restriction mapping analysis has some limited use in analyzing DNA for polymorphisms. If one is looking for a known polymorphism at a site which will change the recognition site for a restriction enzyme, it is possible simply to digest DNA with this restriction enzyme and analyze the relative sizes and numbers of fragments to determine the presence or absence of the polymorphism. (R. K. Saiki et al., Science 230 (1985), 1350-1354). This type of analysis is also useful for determining the presence or absence of gross insertions or deletions, but may not be useful in detecting smaller changes that do not result in a readily distinguishable change in restriction fragment size and/or number. Restriction mapping methods also generally require the use of hybridization techniques which are time consuming and costly.
The large-scale identification of single-nucleotide polymorphisms (SNPs) in the human as well as other model genomes such as yeast and
Arabidopsis thaliana
has been accomplished by methods such as fluorescence-based sequencing (P.-Y. Kwok, Q. et al., Genomics 31 (1996) 123-126), hybridization high-density variation-detection DNA chips (D. G. Wang et al., Science 280 (1998) 1077-1082; E. A. Winzeler et al., Science 281 (1998) 1194-1197), and high performance liquid chromatography (P. A. Underhill et al., Genome Res. 7 (1997) 996-1005; M. Giordano et al., Genomics, 56 (1999) 247-253; R. J. Cho et al., Nature Genet. 23 (1999) 203-207; and M. Cargill et al, Nature Genet. 22 (1999) 231-238). These and other methods have been used to identify thousands of SNPs. For this reason, the development of simple and inexpensive technology for the genotyping of SNPs of individuals (e.g., in a clinical setting) has become of great interest as the ability to discriminate between allelic forms of SNPs is increasingly seen as fundamental to future molecular genetic analysis of disease (N. Risch and K. Merikangas, Science 273 (1996) 1516-1517; F. S. Collins et al., Science 278 (1997) 1580-1581; L. Kruglyak, Nature Genet. 17 (1997) 21-24).
A number of additional methods are available for SNP genotyping such as allele-specific hybridization (R. K. Saiki et al., N. Engl. J. Med. 319 (1988) 537-541; M. Chee et al., Science 274 (1996) 610-614), nick translation PCR (L. G. Lee et al., Nucl. Acids Res. 21 (1993) 3761-3766; K. J. Livaket al., PCR Methods Appl. 4 (1995) 357-362), ligase chain reaction (D. Y. Wu and R. B. Wallace, Genomics 4 (1989) 560-560; D. A. Nickerson et al., Proc. Natl. Acad. Sci. USA 87 (1990) 8923-8927), allele-specific polymerase chain reaction (C. R. Newton et al, Nucl. Acids Res. 17 (1989) 2503-2516; D. Y. Wu et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2757-2760); T
m
-shift genotyping (S. Germer and R. Higuchi, Genome Res. 9 (1999) 72-78), and minisequencing (A. Jalanko et al., Clin. Chem. 38 (1992) 39-43; P. Nyren et al., Anal. Biochem. 208 (1993) 171-175; T. T. Nikiforov et al., Nucl. Acids Res. 22 (1994) 4167-4175; T. Pastinen et al., Clin. Chem. 42 (1996) 1391-1397; G. S. Higgins et al., BioTechniques 23 (1997) 710-714; L. A. Haff and I. P Smirnov, Genome Res. 7 (1997) 378-388; C. A. Piggee et al., J. Chromatogr. A 781 (1997) 367-75; X. Chen et al., Genome Res. 9 (1999) 492-498; and B. Hoogendoom et al., Hum. Genet. 104 (1999) 89-93). The latter method, which is based on the annealing of a primer immediately upstream or downstream from the polymorphic site and its extension by one or more bases in the presence of the appropriate dNTPs and ddNTPs, has become very popular. It has been combined with a variety of techniques for detecting the extension products, including radiolabeling (A. Jalanko et al., Clin. Chem. 38 (1992) 39-43), luminous detection (P. Nyren et al, Anal. Biochem. 208 (1993) 171-175), colorimetric ELISA (T. T. Nikiforov et al., Nucl. Acids Res. 22 (1994) 4167-4175), gel-based fluorescent detection (T. Pastinen et al., Clin. Chem. 42 (1996) 1391-1397), mass spectrometry (G. S. Higgins et al., BioTechniques 23 (1997) 710-714; L. A. Haff and I. P Smimov, Genome Res. 7 (1997) 378-388), capillary electrophoresis (C. A. Piggee et al., J. Chromatogr. A 781 (1997) 367-75), fluorescence polarization (X. Chen et al., Genome Res. 9 (1999) 492-498), and most recently high-performance liquid chromatography (B. Hoogendoom et al., Hum. Genet. 104 (1999) 89-93).
All of the aforementioned genotyping techniques use the polymerase chain reaction as the initial sample pretreatment step. Many of these techniques thus require at least a two-step process to determine the presence of an SNP. Although some of the methods can be done in a single step in a single tube, these techniques require expensive fluorescent dye-labeled oligonucleotide probes (L. G. Lee et al., Nucl. Acids Res. 21 (1993) 3761-3766.; K. J. Livak et al., PCR Methods Appl. 4 (1995) 357-362). Others require additional steps such as hybridization or primer extension. Primer extension also requires prior purification of the PCR product from unincorporated dNTPs and oligonucleotides by either solid-phase extraction or enzymatic treatment with Shrimp Alkaline Phosphatase and Exonuclease I. For these reasons, genotyping is still a far more costly undertaking than identifying the presence of an SNP in the genome. This constitutes a severe limitation in the application of SNPs to genetic studies in the clinic and laboratories.
High-performance liquid chromatography (HPLC) has been used to identify and analyze polymorphisms i
Bozicevic Field & Francis LLP
Brusca John S.
Francis Carol L.
Moran Marjorie A
Stanford University
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