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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

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Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.

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Pyrosequencing protocol requiring a unique biotinylated primer

Jose Luis Royo1 / Manuel Hidalgo Pascual2 / Ana Salinas3 / Francisco Jose Tello4 / Maria del Carmen Rivero5 / Eduardo Ferrero Herrero6 / Luis Miguel Real7 / Agustín Ruiz8









Corresponding author: Jose Luis Royo, Departamento de Genomica Estructural, Neocodex SL, Avda Charles Darwin s/n, Isla de la Cartuja, Sevilla, Spain Phone: +34-955047618, Fax: +34-955047325,

Citation Information: Clinical Chemical Laboratory Medicine. Volume 44, Issue 4, Pages 435–441, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/CCLM.2006.072, April 2006

Publication History

November 18, 2005
January 3, 2006
Published Online:


Background: DNA sequencing has markedly changed the nature of biomedical research. Large-scale sequencing projects have generated several millions of potential polymorphisms widespread in the human genome requiring validation and incorporation into screening panels. As a consequence, high-throughput analysis of these variants in different populations of interest is now the cornerstone of structural genomics. Pyrosequencing is a versatile technique allowing an easy 96-well typing format. However, every polymorphism requires a specific labeled primer to generate a single-stranded DNA fragment containing the region of interest.

Methods: We describe how with an adjusted primer stoichiometry we can standardize the labeling of every amplicon with a single biotinylated universal primer (BM13S).

Results: We circumvent the need for specific biotinylated primers for each single-nucleotide polymorphism (SNP) under study. As an example, we assessed this novel protocol by genotyping three SNPs mapping calpain-10, caveolin-1 and CYP19A1.

Conclusion: The present approach represents an alternative to standard pyrosequencing protocols, since it requires a single biotinylated primer that is suitable for each SNP under study.

Keywords: calpain-10 (CAPN10); caveolin-1 (CAV1); cytochrome P450 (CYP19A1); genotyping; M13; pyrosequencing

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