We recently introduced a generic single nucleotide polymorphism (SNP) genotyping method, termed DASH (dynamic allele-specific hybridization), which entails dynamic tracking of probe (oligonucleotide) to target (PCR product) hybridization as reaction temperature is steadily increased. The reliability of DASH and optimal design rules have not been previously reported. We have now evaluated crudely designed DASH assays (sequences unmodified from genomic DNA) for 89 randomly selected and confirmed SNPs. Accurate genotype assignment was achieved for 89% of these worst-case-scenario assays. Failures were determined to be caused by secondary structures in the target molecule, which could be reliably predicted from thermodynamic theory. Improved design rules were thereby established, and these were tested by redesigning six of the failed DASH assays. This involved reengineering PCR primers to eliminate amplified target sequence secondary structures. This sophisticated design strategy led to complete functional recovery of all six assays, implying that SNPs in most if not all sequence contexts can be effectively scored by DASH. Subsequent empirical support for this inference has been evidenced by ∼30 failure-free DASH assay designs implemented across a range of ongoing genotyping programs. Structured follow-on studies employed standardized assay conditions, and revealed that assay reproducibility (733 duplicated genotypes, six different assays) was as high as 100%, with an assay accuracy (1200 genotypes, three different assays) that exceeded 99.9%. No post-PCR assay failures were encountered. These findings, along with intrinsic low cost and high flexibility, validate DASH as an effective procedure for SNP genotyping.The envisioned benefits of high-throughput single nucleotide polymorphism (SNP) analysis are numerous (Brookes 1999), and several large-scale SNP discovery programs are now underway or have been completed (Taillon-Miller et al. 1998;Wang et al. 1998;Cambien et al. 1999;Cargill et al. 1999;Emahazion et al. 1999;Marshall 1999;Picoult-Newberg et al. 1999). Additionally, a number of SNP databases have been built and are steadily growing in content, that is, HGBASE ; http://hgbase.cgr.ki.se), dbSNP (Smigielski et al. 2000; http://www.ncbi.nlm.nih.gov/ SNP) and the SNP Consortium (TSC) (Marshall 1999; http://snp.cshl.org). In order to fully realize the benefits of such developments, further improvements in SNP genotyping technologies will be required. Critical issues here will include ease of assay design, equipment complexity, assay cost, reliability, accuracy, flexibility, and compatibility with automation. Alternative methods under development in different laboratories possess various advantages and disadvantages, making each suitable for a different range of applications. Arguably, however, standardized and simple assay design in addition to accurate allele determination are perhaps the most important prerequisites for a broadly applicable method. Given these features, further development efforts would enable ...
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The Human Genome Project is expected to yield thousands of single nucleotide polymorphisms (SNPs), which can be used to identify polygenic contributors to disease and ultimately to design individualized prognostic strategies and therapies. Exploiting these, however, will require powerful and automated scoring methods. Current techniques have their roots in allele-specific oligonucleotide hybridization (ASOH) 1 . In its basic form (i.e., hybridization, stringent washing, and signal detection), ASOH is limited by the difficult challenge of defining discriminatory assay conditions. Therefore, newer methods include additional steps furnishing more robust allele scoring. Such procedures include the ligation chain reaction (ASOH plus selective ligation and amplification) 2 , mini-sequencing (ASOH plus a single base extension) 3 , and DNA "chips" (miniaturized ASOH with multiple oligonucleotide probe arrays) 4 . Alternatively, ASOH with single-or dual-labeled probes has been merged with PCR, as in the 5´ exonuclease assay 5 , and with molecular beacons 6 . While effective, these methods also entail considerable optimization efforts and/or costly enzymatic or oligonucleotide labeling steps. We discuss here a new SNP scoring method, dynamic allele-specific hybridization (DASH), that does not suffer these drawbacks.The key to DASH is dynamic heating and coincident monitoring of DNA denaturation. No additional enzymes or reaction steps are involved. DASH therefore retains the simplicity of ASOH, but achieves unambiguous discrimination of all SNP variations using standardized reaction conditions. The assay is conducted in a microtiter plate format compatible with automation, and uses convenient fluorescence signal detection. Method principleAn overview of the DASH procedure is shown in Figure 1. A target sequence is amplified by PCR in which one primer is biotinylated. The biotinylated product strand is bound to a streptavidin-coated microtiter plate well, and the non-biotinylated strand is rinsed away with alkali. An oligonucleotide probe, specific for one allele, is hybridized to the target at low temperature. This forms a duplex DNA region that interacts with a double strand-specific intercalating dye.Upon excitation, the dye emits fluorescence proportional to the amount of doublestranded DNA (probe-target duplex) present. The sample is then steadily heated while fluorescence is continually monitored. A rapid fall in fluorescence indicates the denaturing (or "melting") temperature of the probe-target duplex. When performed under appropriate buffer and dye conditions, a single-base mismatch between the probe and the target results in a dramatic lowering of melting temperature (T m ) that can be easily detected. A model experimentSynthetic 5´-biotinylated oligonucleotide targets were made, representing each of the two alleles of a SNP in the PSEN2 gene with the variant base located in the center. These mock target DNAs and a 50/50 mixture of the two NATURE BIOTECHNOLOGY VOL 17 JANUARY 1999 http://biotech.nature.com 87 (to m...
Fluorescence resonance energy transfer (FRET) is a powerful tool for detecting spatial relationships between macromolecules, one use of which is the tracking of DNA hybridization status. The process involves measuring changes in fluorescence as FRET donor and acceptor moieties are brought closer together or moved farther apart as a result of DNA hybridization/denaturation. In the present study, we introduce a new version of FRET, which we term induced FRET (iFRET), that is ideally suited for melting curve analysis. The innovation entails using a double-strand, DNA-specific intercalating dye (e.g., SYBR Green I) as the FRET donor, with a conventional FRET acceptor affixed to one of the DNA molecules. The SNP genotyping technique dynamic allele specific hybridization (DASH) was used as a platform to compare iFRET to two alternative fluorescence strategies, namely, the use of the intercalating dye alone and the use of a standard FRET pair (fluorescein as donor, 6-rhodamine as acceptor). The iFRET configuration combines the advantages of intercalating dyes, such as high signal strengths and low cost, with maintaining the specificity and multiplex potential afforded by traditional FRET detection systems. Consequently, iFRET represents a fresh and attractive schema for monitoring interactions between DNA molecules.Fluorescence signals may be created by various means to detect DNA hybridization during genotyping and similar assays. The simplest method is to use an intercalating dye (Fig. 1a) that is highly specific for double-stranded DNA. When such a dye (e.g., SYBR Green I) intercalates into a DNA double helix, the dye can fluoresce while exposed to suitable excitatory illumination. Subsequent separation of the DNA strands as part of an assay procedure will cause a release of the dye and consequential loss of fluorescence. This strategy is very inexpensive and yields high levels of fluorescence (Howell et al. 1999), but its inherent limitations include a lack of specificity for any particular duplex and no possibility to create multiplexed assays.A second strategy for the detection of DNA hybridization involves fluorescence resonance energy transfer, or FRET (Fig. 1b). In FRET, a donor fluorophore molecule absorbs excitation energy and delivers this via dipole-dipole interaction to a nearby acceptor fluorophore molecule (Stryer and Haugland 1967), as recently reviewed (Wu and Brand 1994). This process only occurs when the donor and acceptor molecules are sufficiently close to one another. Several different strategies for determining the optimal physical arrangement of the donor and acceptor moieties have been described (Holland et al. 1991;Tyagi and Kramer 1996;Bernard et al. 1998; for review, see Didenko 2001;Solinas et al. 2001). Although FRET enables specification of the target sequence and the potential for multiplexing, this is counterbalanced by the extra expense of physically attaching both the donor and acceptor fluorophores, and a much weaker relative fluorescence intensity.Here, we present an alternative ...
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