High-throughput Single Nucleotide Polymorphism Genotyping Research Articles

Genetik von Vorhofflimmern: seltene Mutationen, häufige Genvarianten und klinische Relevanz?

Atrial fibrillation (AF) is considered the, by far, the most common arrhythmia of man, affecting millions of patients worldwide. The high socio-economic relevance is due to several severe complications and therefore requires profound scientific research in the field of etiology and treatment options. Atrial fibrillation typically occurs in the older patient who often suffers from a number of underlying diseases that act as predisposing factors. That genetics contribute strongly to this rhythm disorder is therefore not evident at a first glance. However, there are a number of investigations that prove familial accumulation for lone AF. Furthermore it is remarkable that many older patients suddenly develop atrial fibrillation without underlying disease, while others remain in sinus rhythm although suffering from a series of risk factors. Considering all this, genetic interference becomes most probable. Therefore in the recent past remarkable endeavours have been ventured to clarify the genetic basis of both lone AF and AF in the context of underlying diseases. For the former, until now four different genetic loci and three disease genes have been identified as causative. Concerning AF in the general population, mainly studies turning the spotlight on single-nucleotide polymorphisms (SNPs) have been applied. It is assumed that SNPs in disease-causing genes are distributed differentially among healthy and diseased individuals. These differences in frequency have been investigated with case-control studies. Up to now six different genes have been found to be associated with AF, including the genes for angiotensin-converting enzyme, angiotensinogen and several cardiac ion channels. Promising new technologies, especially high-throughput SNP genotyping and the genome wide scan for new candidate genes using chip arrays capable of genotyping up to 500 000 SNPs at a time, will multiply the speed to achieve new results. With that the possibility, approaches to optimize existing therapies and to open up new pathways to treat AF.

Rapid Melting Curve Analysis on Monolayered Beads for High-Throughput Genotyping of Single-Nucleotide Polymorphisms

This report describes a rapid solid-phase melting curve analysis method for single-nucleotide polymorphism (SNP) genotyping. The melting curve analysis is based on dynamic allele-specific hybridization (DASH). The DNA duplexes are conjugated on beads that are immobilized on the surface of a microheater chip with integrated heaters and temperature sensors. SNP on PCR products were scored, illustrating the sensitivity and robustness of the system. The method is based on random bead immobilization by microcontact printing. Single-bead detection and multiplexing were performed with a heating rate more than 20 times faster than conventional DASH. Analyses that took more than 15 min could be performed in less that 1 min, enabling ultrarapid SNP analysis. In addition, an array version of the chip was implemented enabling the preparation of an array of bead arrays for high-throughput and rapid SNP genotyping.

Multiplexed SNP genotyping using nanobarcode particle technology

Single-nucleotide polymorphisms (SNP) are the most common form of sequence variation in the human genome. Large-scale studies demand high-throughput SNP genotyping platforms. Here we demonstrate the potential of encoded nanowires for use in a particles-based universal array for high-throughput SNP genotyping. The particles are encoded sub-micron metallic nanorods manufactured by electroplating inert metals such as gold and silver into templates and releasing the resulting striped nanoparticles. The power of this technology is that the particles are intrinsically encoded by virtue of the different reflectivity of adjacent metal stripes, enabling the generation of many thousands of unique encoded substrates. Using SNP found within the cytochrome P450 gene family, and a universal short oligonucleotide ligation strategy, we have demonstrated the simultaneous genotyping of 15 SNP; a format requiring discrimination of 30 encoded nanowires (one per allele). To demonstrate applicability to real-world applications, 160 genotypes were determined from multiplex PCR products from 20 genomic DNA samples.

Pharmacogenomics in Inflammatory Bowel Disease

Inherited variations in the nucleotide sequence of genes influence how individual patients respond to drugs. Most commonly, clinically significant genetic variations consist of single nucleotide polymorphisms (SNPs) within genes that affect drug disposition or drug targets. Up to now, relatively few clinically important examples of inherited traits that affect drug responses have been studied in detail. However, one of the well-characterized examples is highly relevant to inflammatory bowel disease therapeutics, that of thiopurine methyltransferase pharmacogenetics. Individuals with 2 normal alleles of the gene encoding thiopurine methyltransferase metabolize and clear thiopurines such as azathioprine and 6-mercaptopurine rapidly. Individuals with 1 normal and 1 variant allele are intermediate, whereas those with 2 variant alleles clear thiopurines very slowly. Intermediate and slow metabolizers are predisposed to have high active thiopurine drug levels and develop bone marrow suppression. Genomic era technology permits determination of large numbers of SNPs in large numbers of individuals. This capability is allowing the field of pharmacogenomics to become one of the most productive interfaces in translational biomedical research at present. By using high-throughput SNP genotyping, combined with careful phenotypic characterization of disease, pharmacogenomic research carries the potential of identifying individual biomarkers that predict the relative likelihood of benefit or risk from a therapeutic intervention. If this promise can be realized, pharmacogenomics will deliver the opportunity for personalized medicine.

High-Throughput SNP Genotyping by Single-Tube PCR with T m -Shift Primers

Despite many recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, there is still a great need for inexpensive and flexible methods with a reasonable throughput. Here we report substantial modifications and improvements to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans. In this advanced version of the melting temperature (Tm)-shift SNP genotyping method, we attach two GC-rich tails of different lengths to allele-specific PCR primers, such that SNP alleles in genomic DNA samples can be discriminated by the Tms of the PCR products. We have validated 306 SNP assays using this method and achieved a success rate in assay development of greater than 83% under uniform PCR conditions. We have developed a standalone software application to automatically assign genotypes directly from melting curve data. To demonstrate the accuracy of this method, we typed 592 individuals for 6 SNPs and showed a high call rate (>98%) and high accuracy (>99.9%). With this method, 6-10,000 samples can be genotyped per day using a single 384-well real-time thermal cycler with 2-4 standard 384-well PCR instruments.

Use of sequence data from rainbow trout and Atlantic salmon for SNP detection in Pacific salmon

Single nucleotide polymorphisms (SNPs) are a class of genetic markers that are well suited to a broad range of research and management applications. Although advances in genotyping chemistries and analysis methods continue to increase the potential advantages of using SNPs to address molecular ecological questions, the scarcity of available DNA sequence data for most species has limited marker development. As the number and diversity of species being targeted for large-scale sequencing has increased, so has the potential for using sequence from sister taxa for marker development in species of interest. We evaluated the use of Oncorhynchus mykiss and Salmo salar sequence data to identify SNPs in three other species (Oncorhynchus tshawytscha, Oncorhynchus nerka and Oncorhynchus keta). Primers designed based on O. mykiss and S. salar alignments were more successful than primers designed based on Oncorhynchus-only alignments for sequencing target species, presumably due to the much larger number of potential targets available from the former alignments and possibly greater sequence conservation in those targets. In sequencing approximately 89 kb we observed a frequency of 4.30 x 10(-3) SNPs per base pair. Approximately half (53/101) of the subsequently designed validation assays resulted in high-throughput SNP genotyping markers. We speculate that this relatively low conversion rate may reflect the duplicated nature of the salmon genome. Our results suggest that a large number of SNPs could be developed for Pacific salmon using sequence data from other species. While the costs of DNA sequencing are still significant, these must be compared to the costs of using other marker classes for a given application.

A machine to make a future Biotech chronicles

A machine to make a future Biotech chronicles

Improving Quality Control and Workflow Management in High- Throughput Single-Nucleotide Polymorphism Genotyping Environments

Single-nucleotide polymorphism (SNP) genotyping is a fundamental tool in the rapidly growing area of complex diseases and pharmacogenomics. SNP patterns that correlate with disease or response to treatment, respectively, are identified using bioinformatic techniques. We present an integrated laboratory information and management system (LIMS) for our high-throughput TaqMan™-based SNP genotyping platform. Three new client tools (ProjectManager, AssayManager, OrderTool) for our LIMS improve quality control and workflow management. The programs support organizing multiple genotyping experiments as projects, managing reagents with barcodes, and automation of assay ordering. The tools are freely available at our homepage.

High-throughput SNP genotyping by combining exonuclease III, nuclease S1, and acridine-bearing PNA

Recently we reported a precise and straightforward method for genotyping of single nucleotide polymorphisms (SNPs) in double-stranded DNA substrates. The substrates were consecutively treated by two enzymes (exonuclease III and nuclease S1) in the presence of peptide nucleic acid (PNA) probes, and the resultant DNA fragments were analyzed by mass spectroscopy. Here, the selectivity of DNA scission by nuclease S1 has been greatly improved by attaching an acridine to the PNA probes. Modification at the C-terminus was especially effective. The number of DNA fragments formed has been greatly decreased so that double-stranded DNA substrates can be genotyped by using the two enzymes still more easily and precisely.

Microarray-based approach for high-throughput genotyping of single-nucleotide polymorphisms with layer-by-layer dual-color fluorescence hybridization.

Robust, inexpensive, high-throughput methodologies are needed for the analysis of molecular markers. DNA microarrays are widely used in biological and biomedical research as a high-throughput tool. At present, there are two general microarray-based methods for genotyping. One approach involves arraying thousands of short oligonucleotides on glass slides for detection of many single-nucleotide polymorphism (SNP) loci in target DNA (1). This method is particularly well suited for genotyping thousands of markers in a limited number of individuals (2)(3)(4)(5)(6). The other approach involves arraying amplified PCR products on glass slides to detect a few SNPs in a large number of samples. Recently, Flavell et al. (7) developed a tagged microarray marker approach for scoring thousands of samples for a codominant molecular marker. Biotin-terminated allele-specific PCR products are spotted unpurified on streptavidin-coated glass slides and visualized by hybridization of fluorescent detector oligonucleotides to tags attached to the allele-specific PCR primers. The tagged microarray marker approach is an efficient method for genotyping thousands of samples on a glass slide. However, reliable allele-specific PCR conditions are needed. Moreover, the expensive streptavidin-coated glass slides can be stored for only 3 days in phosphate-buffered saline. For many applications in the field of biological research, PCR products are the more convenient probe molecules (8). We have developed a microarray-based method, based on dual-color fluorescence hybridization, for genotyping of SNPs in thousands of individuals (9). The experiment successfully demonstrated that PCR products subjected to dual-color hybridization on …

Estimation of relative allele frequencies of single-nucleotide polymorphisms in different populations by microarray hybridization of pooled DNA

Single-nucleotide polymorphisms (SNPs) are considered useful polymorphic markers for genetic studies of polygenic traits. A new practical approach to high-throughput genotyping of SNPs in a large number of individuals is needed in association study and other studies on relationships between genes and diseases. We have developed an accurate and high-throughput method for determining the allele frequencies by pooling the DNA samples and applying a DNA microarray hybridization analysis. In this method, the combination of the microarray, DNA pooling, probe pair hybridization, and fluorescent ratio analysis solves the dual problems of parallel multiple sample analysis, and parallel multiplex SNP genotyping for association study. Multiple DNA samples are immobilized on a slide and a single hybridization is performed with a pool of allele-specific oligonucleotide probes. The results of this study show that hybridization of microarray from pooled DNA samples can accurately obtain estimates of absolute allele frequencies in a sample pool. This method can also be used to identify differences in allele frequencies in distinct populations. It is amenable to automation and is suitable for immediate utilization for high-throughput genotyping of SNP.

High-throughput Genotyping Methods for Pharmacogenomic Studies

Single nucleotide polymorphisms (SNPs) represent the most common form of sequence variation in human DNA. With the completion of the human genome project, SNP genotyping is being undertaken in a large number of pharmacogenomic studies to identify variants associated with responses to specific drugs. The speed at which the goals of pharmacogenomics will be met depends on the development of a large set of SNP markers and a suite of high throughput genotyping methods. A number of methods for high throughput SNP genotyping have been developed. In most studies, a large number of SNPs must be genotyped in a large number of individuals, so the challenge for most laboratories is to find an affordable method that meets the dual requirements of high accuracy and high throughput. In this review, a number of robust genotyping methods currently in use are described, and estimated instrumentation and reagent costs are compared. Although no single genotyping method will suit all applications, the available technology allows pharmacogenomic studies of nearly any scale to be performed. However, advances are still required in genotyping technology to make whole genome genotyping rapid and cost-effective - a critical component of pharmacogenomic studies.

Genetic risk factors for cerebrovascular disease in children with sickle cell disease: design of a case-control association study and genomewide screen.

BackgroundThe phenotypic heterogeneity of sickle cell disease is likely the result of multiple genetic factors and their interaction with the sickle mutation. High transcranial doppler (TCD) velocities define a subgroup of children with sickle cell disease who are at increased risk for developing ischemic stroke. The genetic factors leading to the development of a high TCD velocity (i.e. cerebrovascular disease) and ultimately to stroke are not well characterized.MethodsWe have designed a case-control association study to elucidate the role of genetic polymorphisms as risk factors for cerebrovascular disease as measured by a high TCD velocity in children with sickle cell disease. The study will consist of two parts: a candidate gene study and a genomewide screen and will be performed in 230 cases and 400 controls. Cases will include 130 patients (TCD ≥ 200 cm/s) randomized in the Stroke Prevention Trial in Sickle Cell Anemia (STOP) study as well as 100 other patients found to have high TCD in STOP II screening. Four hundred sickle cell disease patients with a normal TCD velocity (TCD < 170 cm/s) will be controls. The candidate gene study will involve the analysis of 28 genetic polymorphisms in 20 candidate genes. The polymorphisms include mutations in coagulation factor genes (Factor V, Prothrombin, Fibrinogen, Factor VII, Factor XIII, PAI-1), platelet activation/function (GpIIb/IIIa, GpIb IX-V, GpIa/IIa), vascular reactivity (ACE), endothelial cell function (MTHFR, thrombomodulin, VCAM-1, E-Selectin, L-Selectin, P-Selectin, ICAM-1), inflammation (TNFα), lipid metabolism (Apo A1, Apo E), and cell adhesion (VCAM-1, E-Selectin, L-Selectin, P-Selectin, ICAM-1). We will perform a genomewide screen of validated single nucleotide polymorphisms (SNPs) in pooled DNA samples from 230 cases and 400 controls to study the possible association of additional polymorphisms with the high-risk phenotype. High-throughput SNP genotyping will be performed through MALDI-TOF technology using Sequenom's MassARRAY™ system.DiscussionIt is expected that this study will yield important information on genetic risk factors for the cerebrovascular disease phenotype in sickle cell disease by clarifying the role of candidate genes in the development of high TCD. The genomewide screen for a large number of SNPs may uncover the association of novel polymorphisms with cerebrovascular disease and stroke in sickle cell disease.

A standard protocol for single nucleotide primer extension in the human genome using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Analysis of single nucleotide polymorphisms (SNPs) has become an increasingly important area of research, with numerous applications in medical genetics, population genetics, forensic science, and agricultural biotechnology. Large-scale SNP analyses require the development of methodologies that are economical, flexible, accurate and capable of automation. Primer extension in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is currently emerging as a potential method for high-throughput SNP genotyping. We have evaluated a number of published primer extension methods and refined a simple and robust protocol to analyze human autosomal disease-causing mutations and population genetic markers on the Y-chromosome. Twelve different variant sites were examined, and homozygotes, heterozygotes and hemizygotes were accurately typed. A 100% concordance was observed between SNP genotypes obtained using the MALDI-TOFMS technique and alternative genotyping methods, such as restriction fragment length polymorphism (RFLP) assays and denaturing high-performance liquid chromatography (DHPLC). Since multiple polymorphisms can be detected in single reactions, the method provides a cost-effective approach for SNP analysis. The protocol is also extremely flexible (able to accommodate new markers) and can be adapted to a number of platforms without the use of commercial kits.

Genotyping single nucleotide polymorphisms by mass spectrometry.

In the last decade, the demand for high-throughput DNA analysis methods has dramatically increased, mainly due to the advent of the human genome sequencing project that is now nearing completion. Even though mass spectrometry did not contribute to that project, it is clear that it will have an important role in the post-genome sequencing era, in genomics and proteomics. In genomics, mainly matrix-assisted laser desorption/ionization (MALDI) mass spectrometry will contribute to large-scale single nucleotide polymorphism (SNP) genotyping projects. Here, the development and history of DNA analysis by mass spectrometry is reviewed and put into the context with the requirements of genomics. All major contributions to the field and their status and limitations are described in detail.

High‐Throughput SNP Genotyping

Whole genome approaches using single nucleotide polymorphism (SNP) markers have the potential to transform complex disease genetics and expedite pharmacogenetics research. This has led to a requirement for high-throughput SNP genotyping platforms. Development of a successful high-throughput genotyping platform depends on coupling reliable assay chemistry with an appropriate detection system to maximise efficiency with respect to accuracy, speed and cost. Current technology platforms are able to deliver throughputs in excess of 100 000 genotypes per day, with an accuracy of >99%, at a cost of 20–30 cents per genotype. In order to meet the demands of the coming years, however, genotyping platforms need to deliver throughputs in the order of one million genotypes per day at a cost of only a few cents per genotype. In addition, DNA template requirements must be minimised such that hundreds of thousands of SNPs can be interrogated using a relatively small amount of genomic DNA. As such, it is predicted that the next generation of high-throughput genotyping platforms will exploit large-scale multiplex reactions and solid phase assay detection systems.

High-throughput SNP genotyping with the Masscode system.

QIAGEN Genomics, Inc, has developed the Masscode tagging system for DNA labeling and detection. In this application, the Masscode system is described as applied to high-throughput single-nucleotide polymorphism (SNP) genotyping. The labeling system is based on a small-molecular-weight tag that is covalently attached through a photocleavable linker to a DNA oligonucleotide. The tagged oligonucleotide is used as a primer in an allele-specific PCR SNP discrimination assay. The allele-specific amplicons are differentiated through their Masscode tag assignments. After a photolysis step to cleave the tags from the amplicon, the samples are introduced into a single quadrupole mass spectrometry detection system for analysis. Genotyping determinations are based on the relative proportions of the paired allele tags. The system has a lower limit of detection in the femtomolar range (10(-15) M). At present, 30 different Masscode tags may be used simultaneously in a multiplex fashion to routinely provide more than 40,000 SNP genotyping measurements daily. Further developments will allow for the simultaneous detection of several hundred tags.

Selection and use of SNP markers for animal identification and paternity analysis in U.S. beef cattle.

DNA marker technology represents a promising means for determining the genetic identity and kinship of an animal. Compared with other types of DNA markers, single nucleotide polymorphisms (SNPs) are attractive because they are abundant, genetically stable, and amenable to high-throughput automated analysis. In cattle, the challenge has been to identify a minimal set of SNPs with sufficient power for use in a variety of popular breeds and crossbred populations. This report describes a set of 32 highly informative SNP markers distributed among 18 autosomes and both sex chromosomes. Informativity of these SNPs in U.S. beef cattle populations was estimated from the distribution of allele and genotype frequencies in two panels: one consisting of 96 purebred sires representing 17 popular breeds, and another with 154 purebred American Angus from six herds in four Midwestern states. Based on frequency data from these panels, the estimated probability that two randomly selected, unrelated individuals will possess identical genotypes for all 32 loci was 2.0 x 10(-13) for multi-breed composite populations and 1.9 x 10(-10) for purebred Angus populations. The probability that a randomly chosen candidate sire will be excluded from paternity was estimated to be 99.9% and 99.4% for the same respective populations. The DNA immediately surrounding the 32 target SNPs was sequenced in the 96 sires of the multi-breed panel and found to contain an additional 183 polymorphic sites. Knowledge of these additional sites, together with the 32 target SNPs, allows the design of robust, accurate genotype assays on a variety of high-throughput SNP genotyping platforms.

SNP genotyping with fluorescence polarization detection

When a fluorescent molecule is excited by plane polarized light, the fluorescence emitted is also polarized. The degree of fluorescence polarization (FP) detected, under constant temperature and solvent viscosity, is proportional to the molecular weight of the dye molecule. By monitoring the FP of a fluorescent dye, one can detect significant changes in the molecular weight of the molecule without separation or purification. Because the size of the probe is altered in the course of a number of single nucleotide polymorphism (SNP) genotyping reactions, FP is therefore an excellent detection mechanism for these assays. Indeed, FP detection can be used in SNP genotyping with the primer extension TaqMan((R)) and Invader((R)) assays. Use of FP detection makes it possible to reduce the cost of TaqMan((R)) and Invader((R)) probes by abrogating the need for a fluorescence quencher. Moreover, inexpensive, unpurified, and unlabeled probes are used in the primer extension reaction with FP detection. As an end-point detection mechanism, FP detection is suitable for high-throughput SNP genotyping.

Facile method for automated genotyping of single nucleotide polymorphisms by mass spectrometry.

In the future, analysis of single nucleotide polymorphisms (SNPs) should become a powerful tool for many genetic applications in areas such as association studies, pharmacogenetics and traceability in the agro-alimentary sector. A number of technologies have been developed for high-throughput genotyping of SNPs. Here we present the simplified GOOD assay for SNP genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI). The simplified GOOD assay is a single-tube, purification-free, three-step procedure consisting of PCR, primer extension and phosphodiesterase II digestion followed by mass spectrometric analysis. Due to the application of charge-tag technology, no sample purification is required prior to the otherwise very impurity-sensitive MALDI analysis. The use of methylphosphonate containing primers and ddNTPs or alpha-S-ddNTPs together with a novel DNA polymerase derived from Thermotoga maritima for primer extension allow the fluent preparation of negatively charge-tagged, allele-specific products. A key feature of this polymerase is its preference for ddNTPs and alpha-S-ddNTPs over dNTPs. The simplified GOOD assay was run with automatic liquid handling at the lowest manageable volumes, automatic data acquisition and interpretation. We applied this novel procedure to genotyping SNPs of candidate genes for hypertension and cardiovascular disease.