14 research outputs found
Temperature Switch PCR (TSP): Robust assay design for reliable amplification and genotyping of SNPs
Background Many research and diagnostic applications rely upon the assay of individual single nucleotide polymorphisms (SNPs). Thus, methods to improve the speed and efficiency for single-marker SNP genotyping are highly desirable. Here, we describe the method of temperature-switch PCR (TSP), a biphasic four-primer PCR system with a universal primer design that permits amplification of the target locus in the first phase of thermal cycling before switching to the detection of the alleles. TSP can simplify assay design for a range of commonly used single-marker SNP genotyping methods, and reduce the requirement for individual assay optimization and operator expertise in the deployment of SNP assays. Results We demonstrate the utility of TSP for the rapid construction of robust and convenient endpoint SNP genotyping assays based on allele-specific PCR and high resolution melt analysis by generating a total of 11,232 data points. The TSP assays were performed under standardised reaction conditions, requiring minimal optimization of individual assays. High genotyping accuracy was verified by 100% concordance of TSP genotypes in a blinded study with an independent genotyping method. Conclusion Theoretically, TSP can be directly incorporated into the design of assays for most current single-marker SNP genotyping methods. TSP provides several technological advances for single-marker SNP genotyping including simplified assay design and development, increased assay specificity and genotyping accuracy, and opportunities for assay automation. By reducing the requirement for operator expertise, TSP provides opportunities to deploy a wider range of single-marker SNP genotyping methods in the laboratory. TSP has broad applications and can be deployed in any animal and plant species.Tania Tabone, Diane E Mather and Matthew J Hayde
Detection of 100% of mutations in 124 individuals using a standard UV/Vis microplate reader: a novel concept for mutation scanning
We report the development of a simple and inexpensive assay for the detection of DNA polymorphisms and mutations that is based on the modification of mismatched bases by potassium permanganate. Unlike the chemical cleavage of mismatch assay, which also exploits the reactivity of potassium permanganate to detect genomic variants, the assay we describe here does not require a cleavage manipulation and therefore does not require expensive or toxic chemicals or a separation step, as mismatches are detected using direct optical methods in a microplate format. Studies with individual deoxynucleotides demonstrated that the reactivity with potassium permanganate resulted in a specific colour change. Furthermore, studies with synthetic oligonucleotide heteroduplexes demonstrated that this colour change phenomenon could be applied to detect mismatched bases spectrophotometrically. A collection of plasmids carrying single point mutations in the mouse β-globin promoter region was used as a model system to develop a functional mutation detection assay. Finally, the assay was validated as 100% effective in detecting mismatches in a blinded manner using DNA from patients previously screened for mutations using established techniques, such as sequencing, SSCP and denaturing high-performance liquid chromatography (DHPLC) analysis in DNA fragments up to 300 bp in length
A novel detection method for heteroduplex DNA using carbodiimide-induced interrupted primer extension
Mutations, structural variations, and genome-wide resequencing: Where to from here in our understanding of disease and evolution?
Published in Human Mutation, 2008; 29 (6):886-890 at www.interscience.wiley.comTania Tabon
Spectroscopic scan at 420 nm of a 150 bp PCR product (1 µg) containing two mismatches in the same amplicon after treatment with potassium permanganate (1 mM final concentration) at room temperature
<p><b>Copyright information:</b></p><p>Taken from "Detection of 100% of mutations in 124 individuals using a standard UV/Vis microplate reader: a novel concept for mutation scanning"</p><p>Nucleic Acids Research 2006;34(6):e45-e45.</p><p>Published online 22 Mar 2006</p><p>PMCID:PMC1409816.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The top trace (red) shows the oxidation rate of the mismatched duplex, the middle traces (blue and green) show the oxidation rate of the matched duplex and the bottom trace (brown) shows the oxidation rate of the no-template control sample
Spectroscopic scan at 420 nm of 150 bp PCR products (1 µg), representing all four classes of mismatches, after treatment with potassium permanganate (1 mM final concentration) at room temperature ()
<p><b>Copyright information:</b></p><p>Taken from "Detection of 100% of mutations in 124 individuals using a standard UV/Vis microplate reader: a novel concept for mutation scanning"</p><p>Nucleic Acids Research 2006;34(6):e45-e45.</p><p>Published online 22 Mar 2006</p><p>PMCID:PMC1409816.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> G.T/A.C mismatch at position −57 (). C.T/A.G mismatch at position −43 (). C.C/G.G mismatch at position −76 (). A.A/T.T mismatch at position −2. In each scan, the top trace (red) shows the oxidation rate of the mismatched duplex, the middle trace (blue) shows the oxidation rate of the matched duplex and the bottom trace (brown) shows the oxidation rate of the no-template control sample
Somatic mutations in glioblastoma are associated with methylguanine-DNA methyltransferase methylation
Abnormal Nuclear pore formation triggers apoptosis in the intestinal epithelium of elys-Deficient zebrafish
Background & Aims: Zebrafish mutants generated by ethylnitrosourea-mutagenesis provide a powerful toot for dissecting the genetic regulation of developmental processes, including organogenesis. One zebrafish mutant, "flotte lotte" (flo), displays striking defects in intestinal, liver, pancreas, and eye formation at 78 hours postfertilization (hpf). In this study, we sought to identify the underlying mutated gene in flo and link the generic lesion to its phenotype. Methods: Positional cloning was employed to map the flo mutation. Subcellular characterization of flo embryos was achieved using histology, immunocytochemistry, bromodeoxyuridine incorporation analysis, and confocal and electron microscopy. Results: The molecular lesion in flo is a nonsense mutation in the elys (embryonic large molecule derived from yolk sac) gene, which encodes a severely truncated protein lacking the Elys C-terminal AT-hook DNA binding domain. Recently, the human ELYS protein has been shown to play a critical, and hitherto unsuspected, role in nuclear pore assembly. Although elys messenger RNA (mRNA) is expressed broadly during early zebrafish development, widespread early defects in flo are circumvented by the persistence of maternally expressed elys mRNA until 24 hpf From 72 hpf, elys mRNA expression is restricted to proliferating tissues, including the intestinal epithelium, pancreas, liver, and eye. Cells in these tissues display disrupted nuclear pore formation; ultimately, intestinal epithelial cells undergo apoptosis. Conclusions: Our results demonstrate that Elys regulates digestive organ formation