High‐throughput mutational screening for beta‐thalassemia by single‐nucleotide extension

Galbiati, Silvia; Chiari, Marcella; Macellari, Micol; Ferrari, Maurizio; Cremonesi, Laura; Cretich, Marina

doi:10.1002/elps.200700181

Cited by 6 publications

(3 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Microarrays are also gaining popularity in medical diagnostics as variations in the DNA sequences of individuals may affect how they develop diseases and respond to treatments (van’t Veer et al , 2002). These variations are often observed as differences in a single nucleotide, or single nucleotide polymorphisms (SNP), and SNP detection is being utilized for diagnostic purposes such as evaluating an individual’s risk for a certain disease and for genetic analysis in drug discovery (Galbiati et al , 2007; Syvanen, 2001). In order to take advantage of the microarray technology in SNP studies, it is imperative that the technology be capable of resolving single mismatched hybrids from perfect hybrids which have different binding energies.…”

Section: Introductionmentioning

confidence: 99%

Label-free microarray imaging for direct detection of DNA hybridization and single-nucleotide mismatches

Özkumur

Ahn

Yalçın

et al. 2010

Biosensors and Bioelectronics

Self Cite

View full text Add to dashboard Cite

A novel method is proposed for direct detection of DNA hybridization on microarrays. Optical interferometry is used for label-free sensing of biomolecular accumulation on glass surfaces, enabling dynamic detection of interactions. Capabilities of the presented method are demonstrated by highthroughput sensing of solid-phase hybridization of oligonucleotides. Hybridization of surface immobilized probes with 20 base pair-long target oligonucleotides was detected by comparing the label-free microarray images taken before and after hybridization. Through dynamic data acquisition during denaturation by washing the sample with low ionic concentration buffer, melting of duplexes with a single nucleotide mismatch was distinguished from perfectly matching duplexes with high confidence interval (>97%). The presented technique is simple, robust, and accurate, and eliminates the need of using labels or secondary reagents to monitor the oligonucleotide hybridization.

show abstract

Section: Introductionmentioning

confidence: 99%

Label-free microarray imaging for direct detection of DNA hybridization and single-nucleotide mismatches

Özkumur

Ahn

Yalçın

et al. 2010

Biosensors and Bioelectronics

Self Cite

View full text Add to dashboard Cite

show abstract

“…Mass spectrometry could be used for analysis of the extension products as an alternative to capillary electrophoresis [33], [35] further adding to flexibility. Kobayashi and coauthors [31] and Galbiati et al [34] have previously described single-nucleotide primer extension assays for the detection of groups of mutations very similar to our mutation set. However, both assays require several extension reactions to cover all mutations.…”

Section: Discussionmentioning

confidence: 96%

Efficient Detection of Mediterranean β-Thalassemia Mutations by Multiplex Single-Nucleotide Primer Extension

et al. 2012

View full text Add to dashboard Cite

β-Thalassemias and abnormal hemoglobin variants are among the most common hereditary abnormalities in humans. Molecular characterization of the causative genetic variants is an essential part of the diagnostic process. In geographic areas with high hemoglobinopathy prevalence, such as the Mediterranean region, a limited number of genetic variants are responsible for the majority of hemoglobinopathy cases. Developing reliable, rapid and cost-effective mutation-specific molecular diagnostic assays targeting particular populations greatly facilitates routine hemoglobinopathy investigations. We developed a one-tube single-nucleotide primer extension assay for the detection of eight common Mediterranean β-thalassemia mutations: Codon 5 (-CT); CCT(Pro)->C–, Codon 6 (-A); GAG(Glu)->G-G, Codon 8 (-AA); AAG(Lys)->–G, IVS-I-1 (G->A), IVS-I-6 (T->C), IVS-I-110 (G->A), Codon 39 (C->T), and IVS-II-745 (C->G), as well as the hemoglobin S variant beta 6(A3) Glu>Val. We validated the new assay using previously genotyped samples obtaining 100% agreement between independent genotyping methods. Our approach, applicable in a range of Mediterranean countries, offers a combination of high accuracy and rapidity exploiting standard techniques and widely available equipment. It can be further adapted to particular populations by including/excluding assayed mutations. We facilitate future modifications by providing detailed information on assay design.

show abstract

“…Capillary and microchip electrophoresis have found large application in the detection of point mutation. Single nucleotide extension, sometimes referred to as single base extension 15, amplification refractory mutation system 16 and restriction endonuclease analysis of PCR products (for a review, see 17) are examples of technique relaying on enzymatic reactions followed by electrophoresis. None of these methods was, in our hands, sensitive enough to be applicable to the analysis of minority paternally inherited fetal mutations in the maternal plasma.…”

Section: Introductionmentioning

confidence: 99%

Development of new substrates for high‐sensitive genotyping of minority mutated alleles

et al. 2008

Self Cite

View full text Add to dashboard Cite

An unsurpassed level of sensitivity was reached in the detection of minority mutated alleles. A low-density microarray was printed on a substrate specifically designed to provide an interference effect which amplifies the collection of the light emitted on the support and reinforces the intensity of excitation light. Optimal performance of the array was obtained by maximizing the probe density and the binding efficiency to the target through a polymeric coating made by the adsorption of a copolymer of N,N-dimethylacrylamide (97% of moles), N,N-acryloyloxysuccinimide (2%) and 3-(trimethoxysilyl)propyl methacrylate (1%) synthesized by free radical copolymerization. The new substrate was used in the identification of fetal mutations in the maternal plasma DNA. Amino-modified amplicons from genomic DNA corresponding to the locus of eight beta-thalassemia mutations were immobilized and interrogated with dual-color oligonucleotide targets. Compared with the conventional glass substrates, the new substrate showed a great enhancement of fluorescence signals thanks to the combination of the optics with the highly efficient polymeric coating, allowing specific detection of all mutations. The high sensitivity and selectivity obtained made it possible to develop assays for the identification of paternally inherited mutations on fetal DNA in the maternal plasma in couples at risk for beta-thalassemia.

show abstract

High‐throughput mutational screening for beta‐thalassemia by single‐nucleotide extension

Cited by 6 publications

References 17 publications

Label-free microarray imaging for direct detection of DNA hybridization and single-nucleotide mismatches

Label-free microarray imaging for direct detection of DNA hybridization and single-nucleotide mismatches

Efficient Detection of Mediterranean β-Thalassemia Mutations by Multiplex Single-Nucleotide Primer Extension

Development of new substrates for high‐sensitive genotyping of minority mutated alleles

Contact Info

Product

Resources

About