Apolipoprotein E genotyping: a comparative study between restriction endonuclease mapping and allelic discrimination with the LightCycler

Ballerini, Sabrina; Bellincampi, Lorenza; Bernardini, Sergio; Casciani, Stefania; Motti, Corradino; Cortese, Claudio; Federici, Giorgio

doi:10.1016/s0009-8981(01)00769-0

Cited by 16 publications

(12 citation statements)

References 11 publications

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“…Recently, systems have been developed that integrate microvolume rapid-cycle PCR with fluorometry, offering real-time fluorescent monitoring of the amplification reaction for quantitative PCR and/or characterization of PCR products for rapid genotyping without the need for post-PCR sample manipulation (2,3 ). The LightCycler TM (Roche Molecular Biochemicals) is one such system.…”

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confidence: 99%

Rapid Screening of Multiple β-Globin Gene Mutations by Real-Time PCR on the LightCycler: Application to Carrier Screening and Prenatal Diagnosis of Thalassemia Syndromes

et al. 2003

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Background: Hemoglobinopathies are priority genetic diseases for prevention programs. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis for carrier couples. Methods: As a model, we designed a protocol based on the LightCycler TM technology to screen for a spectrum of ␤-globin gene mutations in the Greek population. Design was facilitated by dual fluorochrome detection and close proximity of many mutations. Three probe sets were capable of screening 95% of ␤-globin gene mutations in the Greek population, including IVSII-745C3 G, HbS, Cd5-CT, Cd6-A, Cd8-AA, IVSI-1G3 A, IVSI-5G3 A, IVSI-6T3 C, IVSI-110G3 A, and Cd39 C3 T. Results: The protocol, standardized by analysis of 100 ␤-thalassemia heterozygotes with known mutations, was 100% reliable in distinguishing wild-type from mutant alleles. Subsequent screening of 100 Greek ␤-thalassemia heterozygotes with unknown mutations found 96 of 100 samples heterozygous for 1 of the 10 mutations, although melting curves were indistinguishable for mutations ⌯bS/Cd6 and IVSI-5/IVSI-1, indicating a need of alternative methods for definitive diagnosis. One sample demonstrating a unique melting curve was characterized by sequencing as Cd8/9؉G. Three samples carried mutations outside the gene region cov-

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confidence: 99%

Rapid Screening of Multiple β-Globin Gene Mutations by Real-Time PCR on the LightCycler: Application to Carrier Screening and Prenatal Diagnosis of Thalassemia Syndromes

et al. 2003

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“…[15] Although 180 causative mutations have been reported for B-thalassemia syndromes, the spectrum of mutations and their frequencies in most populations usually consist of a limited number of common mutations and a slightly larger number of rare mutation [16]. With the aim of rapid DNA analysis, real-time PCR has become an important tool in both research and routine clinical diagnostics [1,2,17,18] Use of the LightCycler facilitates, and allows simultaneous multiple mutation detection through the flexible design of detection probes. Twenty sequenced DNA samples with Bthalassemia mutations and 10 controls (with normal B-globin gene) were included in the study.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, there has been a major transition in the demography of common illnesses over the past years. [2] This change includes a remarkable decline in childhood mortality due to infections and malnutrition in some regions. As a result, many infants with serious genetic disorders such as thalassemia can now survive the early months of life and live long enough to require treatment for their hematologic disorders.…”

Section: Introductionmentioning

confidence: 99%

“…Given the demographic population shifts, thalassemia is at present considered to be a global health problem. [2] In spite of the more than 170 different Bthalassaemia mutations described around the world (http://globin.cse.psu.edu/globinlhtml/huismanltha lsl) [3,4], only five or six of them are responsible for more than 80% of the thalassaemic alleles detected in the different geographic areas with a high prevalence of B-thalassaemia. [5] Thus, in the countries bordering the Mediterranean basin, the major mutations of the B-globin gene are CD-39 (C62434T) and IVS 1-110 (G62388A) [6].…”

Section: Introductionmentioning

confidence: 99%

“…Consequently, there is a need for more rapid, high-throughput assays. Recently, systems have been developed that integrate microvolume rapid-cycle PCR with fluorometry, offering real-time fluorescent monitoring of the amplification reaction for quantitative PCR and/or characterization of PCR products for rapid genotyping without the need for post-PCR sample manipulation [1,2]. The LightCyclerTM (Roche Molecular Biochemicals) is one such system.…”

Section: Introductionmentioning

confidence: 99%

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Rapid screening of β-Globin gene mutations by Real-Time PCR in Egyptian thalassemic children

Gaafar¹,

ELBeshlawy²,

Aziz³

et al. 2008

African Journal of Health Sciences

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SUMMARYThalassemia is one of the most common genetic disorders in Egypt. With the total population of 70 million, there are approximately 600,000 affected individuals and more than 20 million thalassemia carriers. Thalassemia is therefore one of the major health problems in Egypt. B-Thalassemias are priority genetic diseases for prevention programs. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis for carrier couples. Introduction of the real time PCR has made a revolution in the time taken for the PCR reactions. We present a method for the diagnosis of the common mutations of the B-thalassemia in Egyptian children & families. The procedure depends on the real-time PCR using specific fluorescently labeled hybridization probes. The melting temperature for each of the specific probes obtained after the PCR reaction permits the identification of the specific mutation. Genotyping of 20 thalassemic children attending the hematology clinic of the children specialized hospital and 10 controls was done using Real-time PCR and the conventional Amplification Refractory Mutation System (ARMS) technique. Analysis revealed identical results to most of the patients and they were further checked by the sequencing results of the DNA samples. The established method is a robust, fast and straight forward assay that allows the detection of the common B-thalassemia mutations in Egypt. The described LightCycler system protocol can rapidly screen for many B-globin gene mutations.

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APOE genotyping: comparison of three methods

Rihn

Berrahmoune²,

Jouma

et al. 2008

Clin Exp Med

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Apolipoprotein E (apo E) polymorphism is associated with increased risk of cardiovascular and Alzheimer diseases, making its genotyping of potentially predictive value. We developed a rapid, reliable and specific method for determining APOE genotypes by fluorescent resonance energy transfer (FRET) over a high number of samples in a single run using a LightTyper device and dedicated probes. The method, validated with 75 blood samples, was designed to simultaneously detect three common APOE polymorphisms, epsilon(2,) epsilon(3) and epsilon(4), and to identify in a single reaction any of the six following genotypes: epsilon(2)/epsilon(2), epsilon(3)/epsilon(3), epsilon(4)/epsilon(4), epsilon(3)/epsilon(4), epsilon(4)/epsilon(2), epsilon(3)/epsilon(2). The assay involved three phases: (1) DNA extraction, (2) amplification, and (3) melting curve analysis using FRET technique. Briefly, genomic DNA of patients was extracted from total blood. Fragment of APOE was amplified by a first PCR run. Fluorescent labeled probes were added in a second PCR run. FRET genotyping showed following distribution: (1) 1.3% for epsilon(2)/epsilon(2) and epsilon(4)/epsilon(4) homozygotes, (2) 4.0, 6.6 and 14.7% for epsilon(2)/epsilon(4), epsilon(2)/epsilon(3) and epsilon(3)/epsilon(4) heterozygotes, respectively, and (3) 72.0% for epsilon(3)/epsilon(3) homozygotes. Moreover, a careful analysis of the FRET melting curves allowed us to determine the presence of a new polymorphism on the third position of the codon 158 (-AAGCGT-), namely, two nucleotides downstream from the known polymorphism. When the FRET analysis was compared to those obtained by RFLP and sequencing, the presence of this new polymorphism was confirmed only by sequencing thus indicating that RFLP analysis is not always reliable for genotyping.

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Apolipoprotein E genotyping: a comparative study between restriction endonuclease mapping and allelic discrimination with the LightCycler

Cited by 16 publications

References 11 publications

Rapid Screening of Multiple β-Globin Gene Mutations by Real-Time PCR on the LightCycler: Application to Carrier Screening and Prenatal Diagnosis of Thalassemia Syndromes

Rapid Screening of Multiple β-Globin Gene Mutations by Real-Time PCR on the LightCycler: Application to Carrier Screening and Prenatal Diagnosis of Thalassemia Syndromes

Rapid screening of β-Globin gene mutations by Real-Time PCR in Egyptian thalassemic children

APOE genotyping: comparison of three methods

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