Rapid Genotyping of Hemochromatosis Gene Mutations on the LightCycler with Fluorescent Hybridization Probes

Mangasser-Stephan, Kerstin; Tag, Carmen G.; Reiser, Astrid; Gressner, A. M.

doi:10.1093/clinchem/45.10.1875

Cited by 60 publications

(12 citation statements)

References 17 publications

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“…31,39 The latter technique, has found numerous applications, including quantitative PCR, identification and subtyping of infectious agents, and detection of point mutations in oncogenes and in hereditary metabolic diseases. 32,33,40,41 In contrast to many currently available methods for genotyping and mutation detection, including oligonucleotide ligation assay, 42 single-strand confir- mation polymorphism, 43 allele-specific PCR, 44 and PCRrestriction fragment length polymorphism, 45 this technique overcomes the need for time-consuming analytical postamplification steps. Hybridization probes will also detect other sequence variations covered by the probes, which is not the case for restriction fragment length polymorphism analysis.…”

Section: Discussionmentioning

confidence: 99%

“…Measurement of the fluorescence energy is a sensitive monitor for base variations within the target region covered by the probes. 31 Hybridization probes are widely used for the analysis of mutations, for example in the HFE gene in hemochromatosis 32 or in the N-ras gene in cancer. 33 However, the analysis is restricted to see equivalent amounts of the variants or to detect the major variant only.…”

Section: (Am J Pathol 2003 162:737-746)mentioning

confidence: 99%

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One-Step Detection of c-kit Point Mutations Using Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping and Hybridization Probes

Sotlar

Escribano

Landt

et al. 2003

The American Journal of Pathology

194

180

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The prognostic significance of somatic activating codon 816 c-kit mutations in pediatric urticaria pigmentosa has not yet been established in detail. Detection of such mutations in archival paraffin-embedded biopsies is usually hampered by an abundance of surrounding normal cells. Here we describe a method for the selective amplification and specific detection of c-kit mutation Asp816-->Val in complete tissue sections cut from up to 24-year-old paraffin blocks. Peptide nucleic acid-mediated polymerase chain reaction clamping of the wild-type allele was combined with on-line mutation detection using oligonucleotide hybridization probes. In DNA extracted from HMC-1 cells heterozygously carrying the c-kit mutation Asp816-->Val, the one-tube assay allowed specific detection of this mutation in a more than 1000-fold excess of normal background DNA within 1 hour and without the need for additional analytical steps. In a series of 38 cases with pediatric urticaria pigmentosa we detected c-kit codons 815 and 816 mutations in 16 cases. Mutation detection did not correlate with clinical outcome after a mean follow-up of 11.2 years. In conclusion, the procedure described may represent an ideal screening tool for all kinds of clinical applications, using point mutations as markers of, for example, early events in carcinogenesis, circulating metastatic tumor cells, and minimal residual disease.

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Section: Discussionmentioning

confidence: 99%

Section: (Am J Pathol 2003 162:737-746)mentioning

confidence: 99%

One-Step Detection of c-kit Point Mutations Using Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping and Hybridization Probes

Sotlar

Escribano

Landt

et al. 2003

The American Journal of Pathology

194

180

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“…The LightCycler technology uses two fluorescent oligonucleotide probes (one labeled at the 3′ and the other at the 5′ end) with a phosphorylation modification at the 3′ end to prevent the extension. This approach has been introduced as a practical alternative to the conventional PCR for the identification of microorganisms (Woo et al 1998), determination of genotype (Nauck et al 1999) and detection of mutation (Mangasser-Stephan et al 1999). The LUX real-time PCR (Light Upon eXtension real-time PCR) employs a single-labelled primer with a fluorophore at the 3´-end and a corresponding unlabelled primer.…”

Section: Polymerase Chain Reaction (Pcr) and Real-time Pcrmentioning

confidence: 99%

Real time PCR and importance of housekeepings genes for normalization and quantification of mRNA expression in different tissues

Rebouças

Costa

Passos

et al. 2013

Braz. arch. biol. technol.

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The aim of this review was to evaluate the importance of the real-time PCR (qRT-PCR) as a technique for mRNA expression analysis in different tissues. Real-time PCR is widely used for quantification of mRNA levels and is a fundamental tool for basic research, molecular medicine and biotechnology. Genes of references are expressed in a wide variety of tissues and cells with minimal variations in their expression levels, and thus are used to normalize data of mRNA quantification. Software programs, such as geNorm, BestKeeper and NormFinder, have been developed to perform the normalization of data, which help to choose the most stable reference gene. Several genes, such as GAPDH, β-actin, β-tubulin, PGK, UBQ, RPL-19 and 18S rRNA have been suggested as standards in PCR studies, but these genes can have variation in their expression in different tissues, reinforcing the idea that there is no ideal reference gene.

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“…The major problem with conventional mutation scanning methods, however, is that it requires multiple manual steps and takes a very long time to verify detection. An online fluorescence polymerase chain reaction (PCR) detection system and HybProbe chemistry were developed for use in the LightCycler system [5]. Funato et al reported that they had established a rapid and reliable system & Hirokazu Ikeda hiro31@mac.com capable of detecting mutations of the APRT gene using a LightCycler system [6].…”

Section: Introductionmentioning

confidence: 99%

Use of LightCycler mutation analysis to detect type II adenine phosphoribosyltransferase deficiency in two patients with 2,8-dihydroxyadeninuria

et al. 2015

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Recently, a number of methods have been devised for detection of mutations in the field of molecular genetics. The LightCycler system has been used for rapid PCR, while simultaneously quantifying and analyzing the amplification results. We tried to apply the LightCycler system to detect APRT*J allele mutations in two families including two children with 2,8-dihydroxyadenine urolithiasis. The first patient was a 3-year-old girl who presented with left flank pain. The second patient was a 2-year-old girl who presented with complaints of sudden dysuria. The spectrophotometric analysis of the stone fragments of both patients revealed an absorption spectrum for 2,8-DHA. We used the LightCycler system to detect APRT*J mutation. The first patient was homozygous for APRT*J/APRT*J and the second patient was compound heterozygous for APRT*J/APRT*Q0. The genetic diagnosis of APRT deficiency using this system may be useful not only as a diagnostic test for infants with known 2,8-DHA, but also as a screening of infants with a suspicion of urolithiasis. We believed that the LightCycler system still is an important means of identifying APRT*J mutation.

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Rapid Genotyping of Hemochromatosis Gene Mutations on the LightCycler with Fluorescent Hybridization Probes

Cited by 60 publications

References 17 publications

One-Step Detection of c-kit Point Mutations Using Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping and Hybridization Probes

One-Step Detection of c-kit Point Mutations Using Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping and Hybridization Probes

Real time PCR and importance of housekeepings genes for normalization and quantification of mRNA expression in different tissues

Use of LightCycler mutation analysis to detect type II adenine phosphoribosyltransferase deficiency in two patients with 2,8-dihydroxyadeninuria

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