Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-11
2003-06-03
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007900, C435S091100, C435S091200, C536S023100, C536S023500, C536S024310, C536S024330
Reexamination Certificate
active
06573047
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of molecular genetics, particularly the identification and detection of certain nucleotide sequences.
BACKGROUND OF THE INVENTION
Familial clustering of common disorders independent of known risk factors indicates that genetic epidemiology studies may provide important leads to understanding the pathogenesis of many complex diseases and help identify individuals at increased risk. The multifactorial nature of many complex diseases suggests that numerous genes, each with multiple alleles having small to moderate effects, may account for the majority of genetic variation involved in defining risk of common chronic diseases on a population basis. Nucleotide variations that are found in at least one percent of the populations are called single nucleotide polymorphisms, or simply SNPs. SNPs occur in roughly one of every 500 bases. Consequently, some 200,000 SNPs lie within coding regions of genes. Much of the genetic variation between individual humans that contributes to differences in susceptibility to disease is believed to reflect SNP variations in DNA (Risch and Merikangas, 1996). Some SNPs cause or strongly contribute to specific diseases. For example, sickle cell anemia is caused solely by the change of an A to a T in the gene encoding the &bgr;-chain of hemoglobin. There are many reports of positive association of SNPs with complex diseases such as hypertension (Brand et al., 1998), or end-stage renal disease (Yu et al., 1998).
SNPs may also prove to be useful in pharmacogenomics, a new approach to drug design, testing and utilization. Here, the premise is that depending on their genetic makeup, individuals respond differently to particular drugs. On another front, the use of SNPs as biallelic genetic markers offers the promise of rapid, highly automated genotyping.
In existing SNP assays, PCR primers flanking each SNP locus to be interrogated are chosen from publicly available genomic sequence information. In one format, the forward PCR primers are designed such that the nucleotide at the 3′-end of the primer complements the base adjacent to the SNP site. The regions containing the SNP polymorphism are then amplified by PCR and the resulting products are purified prior to a primer extension reaction. The extension reaction uses each PCR product as template and fluorescent dye-labeled dideoxynucleotide triphosphates (ca. 100-fold excess) to identify the base present at each SNP site, i.e., the SNP alleles. Each primer extension experiment requires pair(s) of dye-labeled ddNTPs (e.g., R110-ddTTP and ROX-ddCTP for an A-to-G nucleotide change). The labeled extension products representing the SNP alleles are separated by capillary electrophoresis and detected by laser-induced fluorescence.
Various aspects of existing methods limit their efficacy in analyzing SNPs. For example, some of the following commercially available reagents are used in current SNP assays. Rhodamine dye-labeled terminators are available in 16 dye/base combinations from E.I. DuPont de Nemours & Co. ET-labeled terminators (i.e., Energy-Transfer-labeled terminators) can be purchased from Applied Biosystems (BigDye terminator premix kit), or from Amersham Pharmacia Biotech (DYEnamic ET terminator premix kit). A disadvantage of using the ET-labeled terminators is that they offer no flexibility in the choice of dye/base combinations. Only one set of four ET-labeled terminators is available with a particular ET-label on each base. Moreover, both BigDye and DYEnamic ET terminators use FAM derivatives as donors that provide relatively low signal strengths and spectral purity. Finally, these ET-labeled terminators are not readily available other than as components of kits.
Other problems associated with current SNP assays are as follows: 1) With excitation at one wavelength, single dye-labeled terminators give lower signal intensities than ET-labeled terminators; 2) both single dye-labeled and ET-labeled terminator assays provide no discrimination between the fluorescence emission of the extended target and of the reagents; 3) assays using ET-labeled primers likewise provide no fluorescence emission discrimination between extended and unextended primer; 4) purification is required to remove the large excess of unincorporated labeled-ddNTPs (or labeled primers) to avoid masking of the extended target peak. For research studies, such a purification step can be tolerated, but it needs to be eliminated in high throughput assays.
A template-directed dye-terminator incorporation (TDI) assay, a homogeneous DNA diagnostic solution assay based on fluorescence resonance energy transfer (FRET), has recently been developed. In this assay, amplified genomic DNA fragments containing polymorphic sites are incubated with a 5′-FAM-labeled primer in the presence of allelic acceptor dye-labeled dideoxy terminators (Chen and Kwok, 1997a,b). The FAM-labeled primer is extended one base by the acceptor-labeled terminator specific for the allele present on the template. The reaction mixture is then analyzed for changes in fluorescence intensities without separation. This method detects the intramolecular FRET against a background of intermolecular FRET. A related dye-labeled oligonucleotide ligation (DOL) assay in which a donor dye-labeled common probe is joined to an allele-specific, acceptor dye-labeled probe by DNA ligase has also been developed (Chen and Kwok, 1998, 1999).
There are certain limitations associated with the TDI assay. First, in some instances only an overall fluorescence emission is measured, thus, no multiplexing of SNPs can be performed. Second, the efficiency of FRET is sensitive to the distance between the donor and acceptor in ET primers (Ju et al., 1995; Hung et al. 1997). The primers used in the TDI assay carry the donor dye (FAM) at the 5′-end. Moreover, the primer lengths typically are more than 18 nucleotides long. Therefore, the ET-labeled SNP products are formed with donor-acceptor dye pairs separated by more than 18 bases. This long spacing results in poor ET efficiency and requires that the extended primer be dissociated from the template before detection can occur. Also, with FAM as a donor, the residual donor fluorescence emission is relatively high. Third, this assay involves either awkward calculations that create variant thresholds for different loci (Chen and Kwok, 1997a), or awkward real-time fluorescence detection for each cycle during the primer extension (Chen and Kwok, 1997b). These are not suitable for high throughput assays.
SUMMARY OF THE INVENTION
The present invention provides a variety of methods for analyzing target nucleic acids having a variant site. The invention also provides kits for performing such methods within research, clinical and laboratory settings. The methods generally involve conducting template-dependent primer extension reactions to form an energy transfer labeled extension product if a non-extendible nucleotide provided in the extension reaction mixture is complementary to the nucleotide at the variant site. The extension product includes a donor and acceptor fluorophore that together form a pair. One member of the pair is borne by the primer and the other member by the non-extendible nucleotide. By controlling various parameters such as the position of the fluorophore on the primer and the type of fluorophores utilized, certain methods of the invention can enhance signal strength and purity. Further, certain methods can be performed without the need to dissociate extension product from the target nucleic acid, or to separate other reaction components from the extension product, prior to detection.
More specifically, certain methods for analyzing variant sites in nucleic acids of interest involve hybridizing a primer bearing a first fluorophore to a segment of the target nucleic acid to form a labeled hybrid, wherein the 3′-end of the primer hybridizes to the target nucleic acid immediately adjacent to the variant site. Template-dependent extension of the primer is conducted in the presen
Glazer Alexander N.
Hung Su-Chun
Mathies Richard A.
DNA Sciences, Inc.
Horlick Kenneth R.
Spiegler Alexander H.
Townsend and Townsend / and Crew LLP
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