Clinical Chemistry and Laboratory Medicine (CCLM)
Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)
Editor-in-Chief: Plebani, Mario
Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Schlattmann, Peter / Tate, Jillian R.
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Rank 5 out of 30 in category Medical Laboratory Technology in the 2014 Thomson Reuters Journal Citation Report/Science Edition
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Impact per Publication (IPP) 2015: 2.238
Pyrosequencing protocol requiring a unique biotinylated primer
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
- 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.
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