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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 / Schlattmann, Peter / Tate, Jillian R. / Tsongalis, Gregory J.

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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

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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: 10.1515/CCLM.2006.072, April 2006

Publication History

Received:
November 18, 2005
Accepted:
January 3, 2006
Published Online:
2006-04-07

Abstract

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

Citing Articles

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[2]
Adam C. Smith, Masako Suzuki, Reid Thompson, Sanaa Choufani, Michael J. Higgins, Idy W. Chiu, Jeremy A. Squire, John M. Greally, and Rosanna Weksberg
Genomics, 2012, Volume 99, Number 1, Page 25
[3]
Jose Luis Royo, Manuel Hidalgo, and Agustin Ruiz
Nature Protocols, 2007, Volume 2, Number 7, Page 1734

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