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Results for “single nucleotide polymorphisms”

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At least 595 records · Page 33Linked to original sources
PubMed2000

Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry.

BACKGROUND: We have developed a rapid, high throughput method for single nucleotide polymorphism (SNP) genotyping that employs an oligonucleotide ligation assay (OLA) and flow cytometric analysis of fluorescent microspheres. METHODS: A fluoresceinated oligonucleotide reporter sequence is added to a "capture" probe by OLA. Capture probes are designed to hybridize both to genomic "targets" amplified by polymerase chain reaction and to a separate complementary DNA sequence that has been coupled to a microsphere. These sequences on the capture probes are called "ZipCodes". The OLA-modified capture probes are hybridized to ZipCode complement-coupled microspheres. The use of microspheres with different ratios of red and orange fluorescence makes a multiplexed format possible where many SNPs may be analyzed in a single tube. Flow cytometric analysis of the microspheres simultaneously identifies both the microsphere type and the fluorescent green signal associated with the SNP genotype. RESULTS: Application of this methodology is demonstrated by the multiplexed genotyping of seven CEPH DNA samples for nine SNP markers located near the ApoE locus on chromosome 19. The microsphere-based SNP analysis agreed with genotyping by sequencing in all cases. CONCLUSIONS: Multiplexed SNP genotyping by OLA with flow cytometric analysis of fluorescent microspheres is an accurate and rapid method for the analysis of SNPs.

Chromosomes, Human, Pair 19↗
PubMed2006

Parallel optimization and genotyping of multiple single-nucleotide polymorphism markers by sample pooling approach using cycling-gradient CE with multiple injections.

Increasing importance of single-nucleotide polymorphisms (SNPs) in determination of disease susceptibility or in prediction of therapy response brings attention of many molecular diagnostic laboratories to simple and low-cost SNP genotyping methodologies. We have recently introduced a mutation detection technique based on analysis of homo- and heteroduplex PCR fragments resolved in cycling temperature gradient conditions on a conventional multicapillary-array DNA sequencer. The main advantage of this technique is in its simplicity with no requirement for sample cleanup prior to the analysis. In this report we present a practical application of the technology for genotyping of SNP markers in two separate clinical projects resulting in a combined set of 44 markers screened in over 500 patients. Initially, a design of PCR primers and conditions was performed for each SNP marker. Then, optimization of CE running conditions (limited just to the proper selection of temperature cycling) was performed on pools of 20 DNA samples to increase the probability of having each of the two allele types represented in the sample. After selecting the optimum conditions, screening of markers in patients was performed using a multiple-injection approach for further acceleration of the sample throughput. The rate of successful optimization of experimental conditions without any pre-selection based on the SNP sequence or melting characteristics was 80% from the initial SNP marker candidates. By studying the failed markers, we attempt to identify critical factors enabling successful typing. The presented technique is very useful for low to medium sized SNP genotyping projects mostly applied in pharmacogenomic research as well as in clinical diagnostics. The main advantages include low cost, simple setup and validation of SNP markers.

Electrophoresis, Capillary↗
PubMed2000

Single-nucleotide polymorphism analysis by pyrosequencing.

There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.

Base Sequence↗
PubMed2005

Use of bovine single nucleotide polymorphism markers to verify sample tracking in beef processing.

OBJECTIVE: To determine whether a selected set of 20 single nucleotide polymorphism (SNP) markers derived from beef cattle populations can be used to verify sample tracking in a commercial slaughter facility that processes primarily market (ie, culled) dairy cows. DESIGN: Prospective, blinded validation study. ANIMALS: 165 cows and 3 bulls from 18 states (82% Holstein, 8% other dairy breeds, and 10% beef breeds). PROCEDURE: Blood was collected by venipuncture from randomly chosen animals just prior to slaughter. The purported corresponding liver samples were collected during beef processing, and genotype profiles were obtained for each sample. RESULTS: On the basis of SNP allele frequencies in these cattle, the mean probability that 2 randomly selected individuals would possess identical genotypes at all 20 loci was 4.3 x 10(-8). Thus, the chance of a coincidental genotype match between 2 animals was 1 in 23 million. Genotype profiles confirmed appropriate matching for 152 of the 168 (90.5%) purported blood-liver sample pairs and revealed mismatching for 16 (9.5%) pairs. For the 16 mismatched sample pairs, 33% to 76% of the 20 SNP genotypes did not match (mean, 52%). Discordance that could be attributed to genotyping error was estimated to be < 1% on the basis of results for split samples. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that this selected set of 20 bovine SNP markers is sufficiently informative to verify accuracy of sample tracking in slaughter plants that process beef or dairy cattle. These or similar SNP markers may facilitate high-throughput, DNA-based, traceback programs designed to detect drug residues in tissues, control of animal diseases, and enhance food safety.

Animal Identification Systems↗
PubMed2005

Electrokinetically based approach for single-nucleotide polymorphism discrimination using a microfluidic device.

In this work, we describe and implement an electrokinetic approach for single-nucleotide polymorphism (SNP) discrimination using a PDMS/glass-based microfluidic chip. The technique takes advantage of precise control of the coupled thermal (Joule heating), shear (electroosmosis), and electrical (electrophoresis) energies present at an array of probes afforded by the application of external electrical potentials. Temperature controllers and embedded thermal devices are not required. The chips can be easily and inexpensively fabricated using standard microarray printing methods combined with soft-lithography patterned PDMS fluidics, making these systems easily adaptable to applications using higher density arrays. Extensive numerical simulations of the coupled flow and thermal properties and microscale thermometry experiments are described and used to characterize the in-channel conditions. It was found that optimal conditions for SNP detection occur at a lower temperature on-chip than for typical microarray experiments, thereby revealing the importance of the electrical and shear forces to the overall process. To demonstrate the clinical utility of the technique, the detection of single-base pair mutations in the survival motor neuron gene, associated with the childhood disease spinal muscular atrophy, is conducted.

Cyclic AMP Response Element-Binding Protein↗
PubMed2004

Single nucleotide polymorphism in the hypoxia-inducible factor-1alpha gene in colorectal carcinoma.

Colorectal carcinoma is one of the most common malignancies in the world, and its incidence has increased in recent years. We have reported that expression of hypoxia-inducible factor (HIF)-1alpha correlates with expression of vascular endothelial growth factor (VEGF), tumor stage, lymphatic invasion, venous invasion, and liver metastasis. It has also been reported that a single nucleotide polymorphism (SNP) in exon 12 of HIF-1alpha gene is present in renal cell carcinoma and head and neck squamous cell carcinoma patients. We investigated the C1772T polymorphism in colorectal cancer patients and healthy control subjects to clarify the mechanism of HIF-1alpha activation in colorectal carcinoma. The exon 12 genotype was not associated with sex or age. The distribution of HIF-1alpha genotypes in controls was 89 C/C (89%), 11 C/T (11%), and 0 T/T (0%). The distribution of HIF-1alpha genotypes in colorectal cancer patients was 100 C/C (100%), 0 C/T (0%), and 0 T/T (0%). The difference in genotype distribution between patients and control subjects was significant (p<0.0005). These results suggest that the C1772T polymorphism in HIF-1alpha is not involved in progression or metastasis of colorectal carcinoma.

Adenocarcinoma↗
PubMed2006

Single-nucleotide-polymorphism-specific PCR for quantification and discrimination of Chlamydia pneumoniae genotypes by use of a "locked" nucleic acid.

Single-nucleotide polymorphisms (SNPs) are targets to discriminate intraspecies diversity of bacteria and to correlate a genotype with a potential pathotype. Quantification of polygenotypic populations supports this task for in vitro and in vivo applications. We present a novel assay capable of quantifying mixtures of two genotypes differing by only one SNP.

Bacterial Typing Techniques↗
PubMed2004

A new single nucleotide polymorphism in the chicken pituitary-specific transcription factor (POU1F1) gene associated with growth rate.

The pituitary-specific transcription factor (POU1F1) is a protein which binds to and transactivates promoters of growth hormone (GH), prolactin (PRL) and thyroid-stimulating hormone chain (TSHB)-encoding genes. Ten chicken populations (n = 662), including six Chinese indigenous breeds, White Leghorn, paternal/maternal lines of brown egg layer and a paternal line of broiler, were used to detect single nucleotide polymorphisms in the pituitary-specific transcription factor gene (PIT1) by means of PCR-SSCP. A nucleotide transversion from adenine (A) to thymidine (T) at position 980 of the open reading frame of the PIT1 cDNA (GenBank accession no. AF029892) was identified. This nucleotide transversion results in an alteration of codon 299 from AAC to ATC, which leads to a change from asparagine (Asn) to isoleucine (Ile) in the POU domain of POU1F1. The distribution of allele and genotype frequencies differed significantly between meat-type chickens (higher frequencies of A and A/A) and layer-type chickens (P < 0.01). Another experimental population with growth records was used to evaluate the relationship between this polymorphism and growth rate. The results revealed a positive relationship between genotype A/A and body weight at 8 weeks of age, indicating that the SNP in PIT1 gene is a potential molecular marker for early growth rate in chicken.

Animals↗
PubMed2002

Pyrosequencing: an accurate detection platform for single nucleotide polymorphisms.

Pyrosequencing, a non-electrophoretic method for DNA sequencing, is emerging as a popular platform for analysis of single nucleotide polymorphisms (SNPs). This technology has the advantage of accuracy, ease-of-use, and high flexibility for different applications. Here, we review the methodology and the use of this technique for SNP genotyping, SNP discovery, haplotyping, and allelic frequency studies. In addition, we describe new schemes for template preparation and multiplexing as an effort for cost reduction in large-scale studies.

Alleles↗