Challenges And Current Technologies For Single Nucleotide Polymorphism Genotyping

The specific requirements for SNP genotyping technologies vary strongly with the application area. For example, large-scale genetic association studies performed for the identification of disease susceptibility genes and for the optimization of drug developments require the analysis of many thousands of identified SNP positions in hundreds to thousands of individuals to obtain statistically significant association data. Hence, they demand high-throughput SNP genotyping capable of providing up to 500,000 SNP scores (and more) per day. In addition to the evident need for very low running costs, such high-throughput SNP genotyping technologies must be highly automated and must feature economical, fast, and flexible assay development. In contrast, clinical SNP genotyping in forensic, diagnostic, or therapeutic applications usually does not require such high throughput but strongly demands the highest possible sensitivity, specificity, and reliability of the genotyping results. Moreover, prevention of cross-contamination and sample interchange is pivotal for the use of SNP genotyping results in clinical environments.

Currently applied methods for SNP genotyping are based on four general mechanisms for allele discrimination: primer (or probe) extension, allele-specific PCR, allele-specific hybridization, or structure-specific endo-nuclease cleavage. Although some current methods have considerable potential for high-throughput or clinical genotyping or both, they all have specific restrictions. Methods using primer extension (e.g., pyrosequencing[1] and template-directed dye-terminator incorporation1-2-1) and most chip-based methods require prior target amplification and purification. This causes many individual steps which complicate automation, rise costs, and provide uncontrolled error sources. Despite the fact that many assays based on allele-specific PCR [e.g., ''amplification refractory mutation system'' (ARMS) assay[3] or allele-specific hybridization (e.g., 5'-nuclease assays[4] and molecular beacon assays[5]) are homogeneous, they usually require expensive specialty probes. Moreover, most high-density chip-based methods do not allow for flexible, rapid, and inexpensive implementation of newly identified SNPs. Whereas the Invader assay[6] based on structure-specific enzymatic cleavage overcomes many of the described restrictions, it requires huge amounts of genomic DNA because its sensitivity is about 1 order of magnitude below PCR-based methods. Finally, all methods which employ traditional macroscopic fluorescence techniques—encompassing conventional fluorescence intensity, fluorescence polarization, and fluorescence resonance energy transfer—have only limited potential for miniaturization, an essential step toward cost-efficient high-throughput genotyping.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook


Post a comment