Robot Printing of Reverse Dot Blot Arrays for Human Mutation Detection

Lappin, Stephen; Cahlik, Jeff; Gold, Bert

doi:10.1016/s1525-1578(10)60670-8

Cited by 15 publications

(9 citation statements)

References 17 publications

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“…This method is a simple, rapid and reliable method which allows simultaneous detection of up to 20 different mutations or polymorphisms in a single hybridization assay [32]. There are three common alleles at this locus with 5, 7 and 9 thymidines (T 5 , T 7 , T 9 ) that could influence the processing of exon 9 in the mRNA of CFTR gene.…”

Section: Discussionmentioning

confidence: 99%

“…Reverse Dot Blot reaction was performed according to Lappin et al [32]. In this assay, oligonucleotide probes were synthesized using a C6-amino-linker on the 5' end of the product and were attached onto Biodyne C nylon membrane (from Pall Corporation) activated by 1-Ethyl 3-Dimethyl Aminopropyl Carbodiimide (EDAC).…”

Section: Methodsmentioning

confidence: 99%

“…The hybridization pattern of each PCR product allowed the genotype determination of different subjects (in the intron 8 of CFTR gene). p.Phe508del mutation was also screened for all CF patients using a Reverse dot blot reaction [32]. Table 1.…”

Section: Methodsmentioning

confidence: 99%

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Poly Thymidine Polymorphism and Cystic Fibrosis in a Non-Caucasian Population

et al. 2012

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Abstract.Background: Cystic fibrosis is a monogenic recessive disorder found predominantly in Caucasian population. This disease arises from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In this study we consider poly T polymorphism c.1210-12T [5], c.1210-12T[7], c.1210-12T[9] (T5, T7, T9) in the intron 8 of CFTR gene in normal individuals and cystic fibrosis patients in the north of Iran. Material and methods: 40 CF patients and 40 normal individuals were screened for poly T polymorphism in intron 8 of CFTR gene using Reverse Dot Blot method which was also used to detect p.Phe508del among CF patients. Results: T7 allele is the most prevalent in both normal and CF patients. Its abundance is approximately 75%. T9 and T5 represent approximately 20% and 5% of alleles respectively. T7/ T7 genotype is the most present in both normal and CF patients with 72.5% and 60% prevalence respectively. p.Phe508del was present in 13 CFTR alleles belonging to 7 patients with either homozygote T9/ T9, T7/ T7 or compound heterozygote T7/ T9 genotypes. Conclusion: Contrary to the Caucasians, T7 allele is more frequent in Northern Iranian CF patients. The presence of p.Phe508del and T7 allele in the same framework is reported for the first time in this part of the world. Further investigations of other populations will help to understand whether p.Phe508del arose by selection pressure in this part of the world or was imported from European countries. The abundance of T5, T7, T9 alleles indicates that this polymorphism can be used as one of the informative markers for detection of normal and mutant alleles in prenatal diagnosis or carrier assessment in families with previous history of the disease in regions with high degree of CFTR mutation heterogeneity.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Poly Thymidine Polymorphism and Cystic Fibrosis in a Non-Caucasian Population

et al. 2012

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“…The reverse dot-blot is a widely used tool for routine screening of numerous mutant alleles in the HBA2/HBA1 [Chan et al, 1999, Foglietta et al, 2003] and HBB genes [Cai et al, 1994, Winichagoon et al, 1999. Automated platforms for preparing the reverse dot-blot membranes (strips) have been reported that allow printing of large numbers of strips with higher-density arraying [Lappin et al, 2001] and hence commercialization of the entire process.…”

Section: Methodology Overview: Symphony Of Athousandmentioning

confidence: 99%

Molecular diagnosis of inherited disorders: lessons from hemoglobinopathies

2005

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Hemoglobinopathies constitute a major health problem worldwide, with a high carrier frequency, particularly in certain regions where malaria has been endemic. These disorders are characterized by a vast clinical and hematological phenotypic heterogeneity. Over 1,200 different genetic alterations that affect the DNA sequence of the human alpha-like (HBZ, HBA2, HBA1, and HBQ1) and beta-like (HBE1, HBG2, HBG1, HBD, and HBB) globin genes are mainly responsible for the observed clinical heterogeneity. These mutations, together with detailed information about the resulting phenotype, are documented in the globin locus-specific HbVar database. Family studies and comprehensive hematological analyses provide useful insights for accurately diagnosing thalassemia at the DNA level. For this purpose, numerous techniques can provide accurate, rapid, and cost-effective identification of the underlying genetic defect in affected individuals. The aim of this article is to review the diverse methodological and technical platforms available for the molecular diagnosis of inherited disorders, using thalassemia and hemoglobinopathies as a model. This article also attempts to shed light on issues closely related to thalassemia diagnostics, such as prenatal and preimplantation genetic diagnoses and genetic counseling, for better-quality disease management.

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“…PCR for DNA extracted from a paraffin section, and in situ PCR were performed using degenerative primers to detect cutaneous type HPV (3,4). PCR primers for ß-globin were used as positive control (5). No cutaneous type HPV was detected by either analysis (data not shown).…”

Section: Case Reportmentioning

confidence: 98%