+ Translate
+ Most Popular
The pigeon tick (Argas reflexus): its biology, ecology, and epidemiological aspects
Prevalence of hemoglobin abnormalities in Kebili (Tunisian South)
Lipogranuloma: a preventable complication of dacryocystorhinostomy
Value of basal plasma cortisol assays in the assessment of pituitary-adrenal insufficiency
Bees from the Belgian Congo. The acraensis group of Anthophora
Placing gingival retraction cord
Total serum IgE, allergy skin testing, and the radioallergosorbent test for the diagnosis of allergy in asthmatic children
Acariens plumicoles Analgesoidea parasites des oiseaux du Maroc
Injuries of terminal phalanges of the fingers in children
Biology of flowering and nectar production in pear (Pyrus communis)
Das Reliktvorkommen der Aspisviper (Vipera aspis L.) im Schwarzwald
Hydrological modelling of drained blanket peatland
Pathologic morphology and clinical significance of the anomalous origin of the left circumflex coronary artery from the right coronary artery. General review and autopsy analysis of 30 cases
Cyto genetic analyses of lymphocyte cultures after exposure to calcium cyclamate
Axelrodia riesei, a new characoid fish from Upper Rio Meta in Colombia With remarks concerning the genus Axelrodia and description of a similar, sympatric, Hyphessobrycon-species
Favorable evolution of a case of tuberculosis of pancreas under antibiotic action
RIFM fragrance ingredient safety assessment, Valencene, CAS Registry Number 4630-07-3
Parenteral microemulsions: an overview
Temperate pasture: management for grazing and conservation
Evaluation of a new coprocessed compound based on lactose and maize starch for tablet formulation
Thermal expansion and cracking of three confined water-saturated igneous rocks to 800C
Revision of the genera of the tribe Stigmoderini (Coleoptera: Buprestidae) a discussion of phylogenetic relationships
Anal tuberculosis. Report of a case
Gastric tuberculosis in the past and present
Adaptive responses of the cardiovascular system to prolonged spaceflight conditions: assessment with Holter monitoring

High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution

High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution

Clinical Chemistry 51(10): 1770-1777

ISSN/ISBN: 0009-9147

PMID: 16189378

DOI: 10.1373/clinchem.2005.054924

High-resolution DNA melting analysis with saturation dyes for either mutation scanning of PCR products or genotyping with unlabeled probes has been reported. However, simultaneous PCR product scanning and probe genotyping in the same reaction has not been described. Asymmetric PCR was performed in the presence of unlabeled oligonucleotide probes and a saturating fluorescent DNA dye. High-resolution melting curves for samples in either capillaries (0.3 degrees C/s) or microtiter format (0.1 degrees C/s) were generated in the same containers used for amplification. Melting curves of the factor V Leiden single-nucleotide polymorphism (SNP) and several mutations in exons 10 and 11 of the cystic fibrosis transconductance regulator gene were analyzed for both PCR product and probe melting transitions. Independent verification of genotype for simple SNPs was achieved by either PCR product or probe melting transitions. Two unlabeled probes in one reaction could genotype many sequence variants with simultaneous scanning of the entire PCR product. For example, analysis of both product and probe melting transitions genotyped DeltaF508, DeltaI507, Q493X, I506V, and F508C variants in exon 10 and G551D, G542X, and R553X variants in exon 11. Unbiased hierarchal clustering of the melting transitions identified the specific sequence variants. When DNA melting is performed rapidly and observed at high resolution with saturating DNA dyes, it is possible to scan for mutations and genotype at the same time within a few minutes after amplification. The method is no more complex than PCR and may reduce the need for resequencing.

Please choose payment method:

(PDF emailed within 0-6 h: $19.90)

Accession: 012150567

Download citation: RISBibTeXText

Related references

Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis. Nature Protocols 2(1): 59-66, 2007

Description and validation of high-throughput simultaneous genotyping and mutation scanning by high-resolution melting curve analysis. Human Mutation 30(6): 884-890, 2009

Mutation scanning and genotyping by high-resolution DNA melting analysis in olive germplasm. Genome 52(3): 252-260, 2009

Determining the effectiveness of High Resolution Melting analysis for SNP genotyping and mutation scanning at the TP53 locus. Bmc Genetics 10: 5, 2009

Solution-phase DNA mutation scanning and SNP genotyping by nanoliter melting analysis. Biomedical Microdevices 9(2): 159-166, 2007

Mutation scanning the GJB1 gene with high-resolution melting analysis: implications for mutation scanning of genes for Charcot-Marie-Tooth disease. Clinical Chemistry 53(2): 349-352, 2007

Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor. Bmc Plant Biology 9: 143, 2009

Genotyping of the G1138A mutation of the FGFR3 gene in patients with achondroplasia using high-resolution melting analysis. Clinical Biochemistry 41(3): 162-166, 2008

Sensitivity and specificity of high-resolution melting analysis in screening unknown SNPs and genotyping a known mutation. Animal Science Papers and Reports 28(2): 161-170, 2010

Optimization of high-resolution melting analysis for simultaneous genotyping of two 11β-hydroxysteroid dehydrogenase type 1 gene polymorphisms. Genetic Testing and Molecular Biomarkers 15(1-2): 43-49, 2011

Mutation scanning of the RET protooncogene using high-resolution melting analysis. Clinical Chemistry 52(1): 138-141, 2006

Genotyping of the C742T mutation of the FGFR3 gene causing type 1 thanatophoric dysplasia by high-resolution melting analysis. Journal of Maternal-Fetal and Neonatal Medicine: the Official Journal of the European Association of Perinatal Medicine the Federation of Asia and Oceania Perinatal Societies the International Society of Perinatal Obstetricians 24(1): 186-188, 2011

Validation of high-resolution DNA melting analysis for mutation scanning of the LMNA gene. Clinical Biochemistry 42(9): 892-898, 2009

Mutation scanning of exon 20 of the BRCA1 gene by high-resolution melting curve analysis. Clinical Biochemistry 43(1-2): 178-185, 2010

Comparison of high-resolution melting analysis with denaturing high-performance liquid chromatography for mutation scanning in the ABCA4 gene. Investigative Ophthalmology and Visual Science 51(5): 2615-2619, 2010