Rapid Single-Tube Screening of the C282Y Hemochromatosis Mutation by Real-Time Multiplex Allele-specific PCR without Fluorescent Probes

Abstract Background: An accurate determination of the major HFE mutation (C282Y), which is associated with hereditary hemochromatosis, is important in diagnosis and risk assessment for this disease. We report a single-tube high-throughput PCR method for the detection of C282Y. Methods: We combined three previously described principles: allele-specific PCR, mutagenically separated PCR, and amplicon identification by specific dissociation curves. PCR amplification was performed with fluorescence detection or conventional thermocycler using the same primers, reactant constituents, and cycling protocol. Primer cross-reactions were prevented by deliberate primer:primer and primer:template mismatches. Results: PCR products were identified by their characteristic melting temperatures based on SYBR Green I fluorescence. For each of the 256 random and 17 known HFE C282Y samples, mutant homozygous, wild-type, and heterozygous samples were unequivocally distinguished. Conclusions: This homogeneous assay is rapid, reproducible, does not require fluorescent oligonucleotide probes, and correctly identifies HFE genotypes.

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Single-Tube Genotyping without Oligonucleotide Probes

Genome Research ◽

10.1101/gr.9.1.72 ◽

1999 ◽

Vol 9 (1) ◽

pp. 72-78 ◽

Cited By ~ 2

Author(s):

Søren Germer ◽

Russell Higuchi

Keyword(s):

Pcr Amplification ◽

Oligonucleotide Probes ◽

Nucleotide Polymorphisms ◽

Single Tube ◽

Melting Temperatures ◽

Specific Pcr ◽

Allele Specific ◽

Reverse Primer ◽

Common Reverse Primer ◽

Template Dna

We report the development of a self-contained (homogeneous), single-tube assay for the genotyping of single-nucleotide polymorphisms (SNPs), which does not rely on fluorescent oligonucleotide probes. The method, which we call Tm-shift genotyping, combines allele-specific PCR with the discrimination between amplification products by their melting temperatures (Tm). Two distinct forward primers, each of which contains a 3′-terminal base that corresponds to one of the two SNP allelic variants, are combined with a common reverse primer in a single-tube reaction. A GC-tail is attached to one of the forward allele-specific primers to increase theTm of the amplification product from the corresponding allele. PCR amplification, Tmanalysis, and allele determination of genomic template DNA are carried out on a fluorescence-detecting thermocycler with a dye that fluoresces when bound to dsDNA. We demonstrate the accuracy and reliability ofTm-shift genotyping on 100 samples typed for two SNPs, and recommend it both as a simple and inexpensive diagnostic tool for genotyping medically relevant SNPs and as a high-throughput SNP genotyping method for gene mapping.

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Detection of BRAF mutation in thyroid papillary carcinomas by mutant allele-specific PCR amplification (MASA)

Acta Endocrinologica ◽

10.1530/eje.1.02072 ◽

2006 ◽

Vol 154 (2) ◽

pp. 341-348 ◽

Cited By ~ 75

Author(s):

Maria Rosaria Sapio ◽

Daniela Posca ◽

Giancarlo Troncone ◽

Guido Pettinato ◽

Lucio Palombini ◽

...

Keyword(s):

Dna Sequencing ◽

Braf Mutation ◽

Mutant Allele ◽

Pcr Amplification ◽

Follicular Variant ◽

Direct Dna Sequencing ◽

Specific Pcr ◽

Pcr Products ◽

Allele Specific ◽

Allele Specific Pcr

Objective: The somatic point mutation in the BRAF gene, which results in a valine-to-glutamate substitution at residue 600 (BRAFV600E), is an ideal hallmark of papillary thyroid carcinoma (PTC). However, its prevalence is varyingly reported in different studies, and its expression in the follicular variant PTC is controversial, reducing its potential usefulness as diagnostic marker. Design and methods: We developed an assay based on mutant allele-specific PCR amplification (MASA) to detect BRAF mutation. We compared the sensitivity of MASA, single-strand conformation polymorphism (SSCP) and direct DNA sequencing of PCR products. Then, we used MASA 78 to analyze 78 archival thyroid tissues, including normal samples, follicular adenomas, follicular carcinomas and PTC. Results: The MASA assay proved to be a more sensitive method than SSCP and DNA sequencing of PCR products. BRAF mutation was found by MASA in 19/43 (44.2%) of PTC, including 14/31 (45.2%) classic forms and 5/12 (41.7%) follicular variants. No mutations of BRAF were detected in the normal thyroid tissues, nor in follicular adenomas or follicular carcinomas. No correlation was found between BRAF mutation and clinicopathologic features nor with recurrence during a postoperative follow-up period of 4–11 years. BRAFV600E significantly correlated with absence of node metastasis. Conclusions: BRAFV600E is present in PTC, both in the classic form and in follicular variant with similar prevalence. No correlation was found between BRAF mutation and aggressive clinical behavior. MASA-PCR proved to be a specific, sensitive and reliable method to detect BRAF T1799A in DNA extracted from different sources, including cytologic samples obtained either fresh or from archival glass slides. We propose this method as a useful tool to improve accuracy of preoperative diagnosis identifying PTC from biopsies with indeterminate cytologic findings.

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Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650362 ◽

1996 ◽

Vol 75 (05) ◽

pp. 757-759 ◽

Cited By ~ 11

Author(s):

Rainer Blasczyk ◽

Markus Ritter ◽

Christian Thiede ◽

Jenny Wehling ◽

Günter Hintz ◽

...

Keyword(s):

Direct Sequencing ◽

Activated Protein C ◽

Factor V Leiden ◽

Pcr Amplification ◽

Factor V ◽

Specific Pcr ◽

Pcr Rflp ◽

Allele Specific ◽

Specific Amplification ◽

Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

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